Antibodies and molecules derived therefrom that bind to steap-1 proteins

ABSTRACT

Antibodies and molecules derived therefrom that bind to novel STEAP-1 protein, and variants thereof, are described wherein STEAP-1 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, STEAP-1 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The STEAP-1 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with STEAP-1 can be used in active or passive immunization

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/136,897 filed Aug. 12, 2011, which is aDivisional of U.S. patent application Ser. No. 11/587,197, filed Jul.17, 2008, now issued as U.S. Patent No. 8,008,442, which is a NationalPhase application under 35 U.S.C. §371 of International Application No.PCT/US04/12625, filed on Apr. 22, 2004, the entire disclosures of whichare expressly incorporated by reference herein.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392020601SeqList.txt,date recorded: Jan. 23, 2015, size: 226 KB).

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

FIELD OF THE INVENTION

The invention described herein relates to antibodies, as well as bindingfragments thereof and molecules engineered therefrom, that bindproteins, termed STEAP-1. The invention further relates to diagnostic,prognostic, prophylactic and therapeutic methods and compositions usefulin the treatment of cancers that express STEAP-1.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, as reported by the American Cancer Society,cancer causes the death of well over a half-million people annually,with over 1.2 million new cases diagnosed per year. While deaths fromheart disease have been declining significantly, those resulting fromcancer generally are on the rise. In the early part of the next century,cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,ovary, and bladder represent the primary causes of cancer death. Theseand virtually all other carcinomas share a common lethal feature. Withvery few exceptions, metastatic disease from a carcinoma is fatal.Moreover, even for those cancer patients who initially survive theirprimary cancers, common experience has shown that their lives aredramatically altered. Many cancer patients experience strong anxietiesdriven by the awareness of the potential for recurrence or treatmentfailure. Many cancer patients experience physical debilitationsfollowing treatment. Furthermore, many cancer patients experience arecurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 30,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,however its specificity and general utility is widely regarded aslacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein et al., 1997, Nat. Med.3:402). More recently identified prostate cancer markers include PCTA-1(Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res1996 Sep. 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad SciUSA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA)(Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adultmalignancies. Once adenomas reach a diameter of 2 to 3 cm, malignantpotential exists. In the adult, the two principal malignant renal tumorsare renal cell adenocarcinoma and transitional cell carcinoma of therenal pelvis or ureter. The incidence of renal cell adenocarcinoma isestimated at more than 29,000 cases in the United States, and more than11,600 patients died of this disease in 1998. Transitional cellcarcinoma is less frequent, with an incidence of approximately 500 casesper year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma formany decades. Until recently, metastatic disease has been refractory toany systemic therapy. With recent developments in systemic therapies,particularly immunotherapies, metastatic renal cell carcinoma may beapproached aggressively in appropriate patients with a possibility ofdurable responses. Nevertheless, there is a remaining need for effectivetherapies for these patients.

Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there was an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and eight per 100,000 in women. The historicmale/female ratio of 3:1 may be decreasing related to smoking patternsin women. There were an estimated 11,000 deaths from bladder cancer in1998 (7,800 in men and 3,900 in women). Bladder cancer incidence andmortality strongly increase with age and will be an increasing problemas the population becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managedwith a combination of transurethral resection of the bladder (TUR) andintravesical chemotherapy or immunotherapy. The multifocal and recurrentnature of bladder cancer points out the limitations of TUR. Mostmuscle-invasive cancers are not cured by TUR alone. Radical cystectomyand urinary diversion is the most effective means to eliminate thecancer but carry an undeniable impact on urinary and sexual function.There continues to be a significant need for treatment modalities thatare beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in theUnited States, including 93,800 cases of colon cancer and 36,400 ofrectal cancer. Colorectal cancers are the third most common cancers inmen and women. Incidence rates declined significantly during 1992-1996(−2.1% per year). Research suggests that these declines have been due toincreased screening and polyp removal, preventing progression of polypsto invasive cancers. There were an estimated 56,300 deaths (47,700 fromcolon cancer, 8,600 from rectal cancer) in 2000, accounting for about11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectalcancer, and for cancers that have not spread, it is frequently curative.Chemotherapy, or chemotherapy plus radiation, is given before or aftersurgery to most patients whose cancer has deeply perforated the bowelwall or has spread to the lymph nodes. A permanent colostomy (creationof an abdominal opening for elimination of body wastes) is occasionallyneeded for colon cancer and is infrequently required for rectal cancer.There continues to be a need for effective diagnostic and treatmentmodalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancerin 2000, accounting for 14% of all U.S. cancer diagnoses. The incidencerate of lung and bronchial cancer is declining significantly in men,from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s,the rate of increase among women began to slow. In 1996, the incidencerate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000,accounting for 28% of all cancer deaths. During 1992-1996, mortalityfrom lung cancer declined significantly among men (−1.7% per year) whilerates for women were still significantly increasing (0.9% per year).Since 1987, more women have died each year of lung cancer than breastcancer, which, for over 40 years, was the major cause of cancer death inwomen. Decreasing lung cancer incidence and mortality rates most likelyresulted from decreased smoking rates over the previous 30 years;however, decreasing smoking patterns among women lag behind those ofmen. Of concern, although the declines in adult tobacco use have slowed,tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by thetype and stage of the cancer and include surgery, radiation therapy, andchemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expectedto occur among women in the United States during 2000. Additionally,about 1,400 new cases of breast cancer were expected to be diagnosed inmen in 2000. After increasing about 4% per year in the 1980s, breastcancer incidence rates in women have leveled off in the 1990s to about110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women,400 men) in 2000 due to breast cancer. Breast cancer ranks second amongcancer deaths in women. According to the most recent data, mortalityrates declined significantly during 1992-1996 with the largest decreasesin younger women, both white and black. These decreases were probablythe result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient'spreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy; chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amountsof surrounding normal breast tissue may prevent the local recurrence ofthe DCIS. Radiation to the breast and/or tamoxifen may reduce the chanceof DCIS occurring in the remaining breast tissue. This is importantbecause DCIS, if left untreated, may develop into invasive breastcancer. Nevertheless, there are serious side effects or sequelae tothese treatments. There is, therefore, a need for efficacious breastcancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the UnitedStates in 2000. It accounts for 4% of all cancers among women and rankssecond among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

Surgery, radiation therapy, and chemotherapy are treatment options forovarian cancer. Surgery usually includes the removal of one or bothovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus(hysterectomy). In some very early tumors, only the involved ovary willbe removed, especially in young women who wish to have children. Inadvanced disease, an attempt is made to remove all intra-abdominaldisease to enhance the effect of chemotherapy. There continues to be animportant need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in theUnited States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options forpancreatic cancer. These treatment options can extend survival and/orrelieve symptoms in many patients but are not likely to produce a curefor most. There is a significant need for additional therapeutic anddiagnostic options for cancers. These include the use of antibodies,vaccines, and small molecules as treatment modalities. Additionally,there is also a need to use these modilities as research tools todiagnose, detect, monitor, and further the state of the art in all areasof cancer treatment and studies.

SUMMARY OF THE INVENTION

The invention provides antibodies as well as binding fragments thereofand molecules engineered therefrom, that bind to STEAP-1 proteins andpolypeptide fragments of STEAP-1 proteins. As used herein, the termSTEAP-1 is synonamous with 8P1D4. The invention comprises polyclonal andmonoclonal antibodies, murine and other mammalian antibodies, chimericantibodies, humanized and fully human antibodies, and antibodies labeledwith a detectable marker or therapeutic agent. In certain embodiments,there is a proviso that the entire nucleic acid sequence of FIG. 2 isnot encoded and/or the entire amino acid sequence of FIG. 2 is notprepared. In certain embodiments, the entire nucleic acid sequence ofFIG. 2 is encoded and/or the entire amino acid sequence of FIG. 2 isprepared, either of which are in respective human unit dose forms.

The invention further provides methods for detecting the presence andstatus of STEAP-1 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express STEAP-1.An embodiment of this invention provides methods for monitoring STEAP-1gene products in a tissue or hematology sample having or suspected ofhaving some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express STEAP-1such as cancers of tissues listed in Table I, including therapies aimedat inhibiting the transcription, translation, processing or function ofSTEAP-1 as well as cancer vaccines. In one aspect, the inventionprovides compositions, and methods comprising them, for treating acancer that expresses STEAP-1 in a human subject wherein the compositioncomprises a carrier suitable for human use and a human unit dose of oneor more than one agent that inhibits the production or function ofSTEAP-1. Preferably, the carrier is a uniquely human carrier. In anotheraspect of the invention, the agent is a moiety that is immunoreactivewith STEAP-1 protein. Non-limiting examples of such moieties include,but are not limited to, antibodies (such as single chain, monoclonal,polyclonal, humanized, chimeric, or human antibodies), functionalequivalents thereof (whether naturally occurring or synthetic), andcombinations thereof. The antibodies can be conjugated to a diagnosticor therapeutic moiety. In another aspect, the agent is a small moleculeas defined herein.

In another aspect, the agent comprises one or more than one peptidewhich comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLAclass I molecule in a human to elicit a CTL response to STEAP-1 and/orone or more than one peptide which comprises a helper T lymphocyte (HTL)epitope which binds an HLA class II molecule in a human to elicit an HTLresponse. The peptides of the invention may be on the same or on one ormore separate polypeptide molecules. In a further aspect of theinvention, the agent comprises one or more than one nucleic acidmolecule that expresses one or more than one of the CTL or HTL responsestimulating peptides as described above. In yet another aspect of theinvention, the one or more than one nucleic acid molecule may express amoiety that is immunologically reactive with STEAP-1 as described above.The one or more than one nucleic acid molecule may also be, or encodes,a molecule that inhibits production of STEAP-1. Non-limiting examples ofsuch molecules include, but are not limited to, those complementary to anucleotide sequence essential for production of STEAP-1 (e.g. antisensesequences or molecules that form a triple helix with a nucleotide doublehelix essential for STEAP-1 production) or a ribozyme effective to lyseSTEAP-1 mRNA.

Another embodiment of the invention is antibody epitopes, which comprisea peptide regions, or an oligonucleotide encoding the peptide region,that has one two, three, four, or five of the following characteristics:

i) a peptide region of at least 5 amino acids of a particular peptide ofFIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Hydrophilicity profile of FIG. 5;

ii) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equalto 0.0, in the Hydropathicity profile of FIG. 6;

iii) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Percent Accessible Residues profile of FIG. 7;

iv) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Average Flexibility profile of FIG. 8; or

v) a peptide region of at least 5 amino acids of a particular peptide ofFIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Beta-tum profile of FIG. 9.

The present invention also relates to a gene, designated STEAP-1, thathas been found to be over-expressed in the cancer(s) listed in Table I.Northern blot expression analysis of STEAP-1 gene expression in normaltissues shows a restricted expression pattern in adult tissues. Thenucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences ofSTEAP-1 are provided. The tissue-related profile of STEAP-1 in normaladult tissues, combined with the over-expression observed in the tissueslisted in Table I, shows that STEAP-1 is aberrantly over-expressed in atleast some cancers, and thus serves as a useful diagnostic,prophylactic, prognostic, and/or therapeutic target for cancers of thetissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary toall or part of the STEAP-1 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encodingSTEAP-1-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80,85, 90, 95, 100 or more than 100 contiguous amino acids of aSTEAP-1-related protein, as well as the peptides/proteins themselves;DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides oroligonucleotides complementary or having at least a 90% homology to theSTEAP-1 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides that hybridize to the STEAP-1 genes, mRNAs, or toSTEAP-1-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding STEAP-1. Recombinant DNA moleculescontaining STEAP-1 polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of STEAP-1gene products are also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The STEAP-1 SSH sequence of 436 nucleotides.

FIGS. 2A-2Q. The cDNA and amino acid sequence of STEAP-1 variant 1 (alsocalled “STEAP-1 v.1” or “STEAP-1 variant 1”) is shown in FIG. 2A. Thestart methionine is underlined. The open reading frame extends fromnucleic acid 66-1085 including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 2 (also called“STEAP-1 v.2”) is shown in FIG. 2B. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 3 (also called“STEAP-1 v.3”) is shown in FIG. 2C. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-944including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 4 (also called“STEAP-1 v.4”) is shown in FIG. 2D. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 5 (also called“STEAP-1 v.5”) is shown in FIG. 2E. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 6 (also called“STEAP-1 v.6”) is shown in FIG. 2F. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 7 (also called“STEAP-1 v.7”) is shown in FIG. 2G. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 8 (also called“STEAP-1 v.8”) is shown in FIG. 2H. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 9 (also called“STEAP-1 v.9”) is shown in FIG. 2I. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 10 (also called“STEAP-1 v.10”) is shown in FIG. 2J. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 11 (also called“STEAP-1 v.11”) is shown in FIG. 2K. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 12 (also called“STEAP-1 v.12”) is shown in FIG. 2L. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 13 (also called“STEAP-1 v.13”) is shown in FIG. 2M. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 14 (also called“STEAP-1 v.14”) is shown in FIG. 2N. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 15 (also called“STEAP-1 v.15”) is shown in FIG. 20. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 16 (also called“STEAP-1 v.16”) is shown in FIG. 2P. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon.

The cDNA and amino acid sequence of STEAP-1 variant 17 (also called“STEAP-1 v.17”) is shown in FIG. 2Q. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 96-872including the stop codon. As used herein, a reference to STEAP-1includes all variants thereof, including those shown in FIGS. 10, 11and/or 12 unless the context clearly indicates otherwise.

FIGS. 3A-3D. Amino acid sequence of STEAP-1 v.1 is shown in FIG. 3A; ithas 339 amino acids.

The amino acid sequence of STEAP-1 v.2 is shown in FIG. 3B; it has 258amino acids.

The amino acid sequence of STEAP-1 v.3 is shown in FIG. 3C; it has 282amino acids.

The amino acid sequence of STEAP-1 v.4 is shown in FIG. 3D; it has 258amino acids. As used herein, a reference to STEAP-1 includes allvariants thereof, including those shown in FIGS. 10, 11 and/or 12 unlessthe context clearly indicates otherwise.

FIGS. 4A-4C. FIG. 4A. The amino acid sequence alignment of STEAP-1 v.1with mouse TNFa-induced adipose-related protein (gi/16905133). FIG. 4B.The amino acid sequence alignment of STEAP-1 v.1 with rat pHyde protein(gi/21717655/). FIG. 4C. Shows Homology of STEAP-1 to mouse sixtransmembrane epithelial antigen of the prostate (gi|20820492|).

FIGS. 5A & 5B. Hydrophilicity amino acid profile of STEAP-1 variant 1(FIG. 5A). Hydrophilicity amino acid profile of STEAP-1 variant 3 (FIG.5B), determined by computer algorithm sequence analysis using the methodof Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci.U.S.A. 78:3824-3828) accessed on the ProtScale website located on theworld wide web URL ch/cgi-bin/protscale.pl) through the ExPasy molecularbiology server.

FIGS. 6A & 6B. (FIG. 6A). Hydropathicity amino acid profile of STEAP-1variant 1. (FIG. 6B). Hydropathicity amino acid profile of STEAP-1variant 3, determined by computer algorithm sequence analysis using themethod of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J. Mol.Biol. 157:105-132) accessed on the ProtScale website located on theworld wide web URL expasy.ch/cgi-bin/protscale.pl) through the ExPasymolecular biology server.

FIGS. 7A & 7B. (FIG. 7A). Percent accessible residues amino acid profileof STEAP-1 variant 1. (FIG. 7B). Percent accessible residues amino acidprofile of STEAP-1 variant 3, determined by computer algorithm sequenceanalysis using the method of Janin (Janin J., 1979 Nature 277:491-492)accessed on the ProtScale website located on the world wide web URLexpasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biologyserver.

FIGS. 8A & 8B. (FIG. 8A). Average flexibility amino acid profile ofSTEAP-1 variant 1. (FIG. 8B). Average flexibility amino acid profile ofSTEAP-1 variant 3, determined by computer algorithm sequence analysisusing the method of Bhaskaran and Ponnuswamy (Bhaskaran R., andPonnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessedon the ProtScale website located on the world wide web URLexpasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biologyserver.

FIGS. 9A & 9B. (FIG. 9A). Beta-turn amino acid profile of STEAP-1variant 1. (FIG. 9B). Beta-turn amino acid profile of STEAP-1 variant 3,determined by computer algorithm sequence analysis using the method ofDeleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering1:289-294) accessed on the ProtScale website located on the world wideweb URL expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecularbiology server.

FIG. 10. Schematic alignment of SNP variants of STEAP-1. VariantsSTEAP-1 v.4 through v.17 are variants with single nucleotide differencesas compared to STEAP-1 v.2. Though these SNP variants are shownseparately, they could also occur in any combinations and in anytranscript variants that contains the base pairs, e.g., STEAP-1 v.1 andv.3. Numbers correspond to those of STEAP-1 v.2. Black box shows thesame sequence as STEAP-1 v.2. SNPs are indicated above the box.

FIG. 11. Exon compositions of transcript variants of STEAP-1. Thisfigure shows the structure of the transcript variants without poly Atail. Variants STEAP-1 v.1, v.2 and v.3 are transcript variants thatshare the same exons 2 and 3. The first exon of STEAP-1 v.1 is 30 basesshorter at 5′ end than the first exons of the other two transcriptvariants. The fourth exon of STEAP-1 v.2 is the same as the combinedexon 4, intron 4 and exon 5 of STEAP-1 v.1. Compared with STEAP-1 v.1,variant STEAP-1 v.3 has an additional exon spliced out from intron 4 ofSTEAP-1 v.1. Lengths of introns and exons are not proportional.

FIG. 12. Schematic alignment of protein variants of STEAP-1. Proteinvariants correspond to nucleotide variants. Nucleotide variants STEAP-1v.5 through v.17 in FIG. 10 code for the same protein as STEAP-1 v.2.Proteins translated from transcript variants STEAP-1 v.1 and v.3 asshown in FIG. 11 may contain amino acid F (Phe) or L (Leu) at position169. Single amino acid differences were indicated above the boxes. Blackboxes represent the same sequence as STEAP-1 v.1. Boxes with differentpatterns of filling show different sequences. Numbers underneath the boxcorrespond to STEAP-1 v.1.

FIGS. 13A-13C. Secondary structure and transmembrane domains predictionfor STEAP-1 protein variants. The secondary structure of STEAP-1 proteinvariants 1 (SEQ ID NO: 46), 2 (SEQ ID NO: 47), and 3 (SEQ ID NO: 48);(FIGS. 13 a-13 c, respectively) were predicted using theHNN—Hierarchical Neural Network method (Guermeur, 1997,http:/pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessedfrom the ExPasy molecular biology server located on the World Wide Webat (.expasy.ch/tools/). This method predicts the presence and locationof alpha helices, extended strands, and random coils from the primaryprotein sequence. The percent of the protein in a given secondarystructure is also listed. FIGS. 13D, 13F, and 13H: Schematicrepresentations of the probability of existence of transmembrane regionsand orientation of STEAP-1 variant 1-3, (FIGS. 13( d), 13(f) and 13(h)respectively, based on the TMpred algorithm of Hofmann and Stoffel whichutilizes TMBASE (K. Hofmann, W. Stoffel. TMBASE—A database of membranespanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). FIGS.13E, 13G, and 13I: Schematic representations of the probability of theexistence of transmembrane regions and the extracellular andintracellular orientation of STEAP-1 variants 1-3, FIGS. 13( e), 13(g),and 13(i) respectively, based on the TMHMM algorithm of Sonnhammer, vonHeijne, and Krogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and AndersKrogh: A hidden Markov model for predicting transmembrane helices inprotein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systemsfor Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major,R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAA, Press,1998). The TMpred and TMHMM algorithms are accessed from the ExPasymolecular biology server located on the World Wide Web at(.expasy.ch/tools/).

FIGS. 14A-14F. FIG. 14A. Expression of STEAP-1 in stomach cancer patientspecimen. RNA was extracted from normal stomach (N) and from 10different stomach cancer patient specimens (T). Northern blot with 10 μgof total RNA/lane was probed with STEAP-1 sequence. Results show strongexpression of an approximately 1.6 kb STEAP-1 in the stomach tumortissues. The lower panel represents ethidium bromide staining of theblot showing quality of the RNA samples. FIG. 14B. STEAP-1 expression inrectum cancer patient tissues. RNA was extracted from normal rectum (N),rectum cancer patient tumors (T), and rectum cancer metastasis (M).Northern blots with 10 μg of total RNA were probed with the STEAP-1sequence. Results show strong expression of STEAP-1 in the rectum cancerpatient tissues. The lower panel represents ethidium bromide staining ofthe blot showing quality of the RNA samples. FIG. 14C. Expression ofSTEAP-1 in human umbilical vein endothelial cells (HUVEC). First strandcDNA was prepared from HUVEC cells, LAPC-4AD and LAPC-9AD prostatecancer xenografts, as well as from human brain tissues. Normalizationwas performed by PCR using primers to actin and GAPDH. Semi-quantitativePCR, using primers to STEAP-1, was performed at 27 and 30 cycles ofamplification (A). As a control, PCR using primers to actin is shown in(B). Results show strong expression of STEAP-1 in HUVEC cells similar tothe expression detected in prostate cancer xenograft tissues. Expressionof STEAP-1 in HUVEC cells indicates that targeting STEAP-1 may alsotarget endothelial cells of the neovasculature of the tumors. FIG. 14Dand FIG. 14E. STEAP-1 Expression in Normal and Cancer Tissues. Firststrand cDNA was prepared from normal tissues (bladder, brain, heart,kidney, liver, lung, prostate, spleen, skeletal muscle, testis,pancreas, colon and stomach), and from pools of patient cancer specimens(prostate cancer pool, bladder cancer pool, kidney cancer pool, coloncancer pool, lung cancer pool, ovary cancer pool, breast cancer pool,cancer metastasis pool, pancreas cancer pool, prostate cancer xenograftpool, and prostate metastasis to lymph node pool. Normalization wasperformed by PCR using primers to actin. Semi-quantitative PCR, usingprimers to STEAP-1, was performed at 26 and 30 cycles of amplification.In (FIG. 14D) picture of the RT-PCR agarose gel is shown. In (FIG. 14E)PCR products were quantitated using the AlphaImager software. Resultsshow strong of expression of STEAP-1 in normal prostate amongst all thenormal tissues tested. Upregulation of STEAP-1 expression was detectedin prostate cancer pool, bladder cancer pool, kidney cancer pool, coloncancer pool, lung cancer pool, ovary cancer pool, breast cancer pool,and pancreatic cancer pool. Strong expression of STEAP-1 was detected incancer metastasis pool, prostate cancer xenograft pool, and prostatemetastasis to lymph node. FIG. 14F: STEAP-1 Expression in lymphomapatient specimens. First strand cDNA was prepared from a panel oflymphoma patient specimens. Normalization was performed by PCR usingprimers to actin. Semi-quantitative PCR, using primers to STEAP-1, wasperformed at 26 and 30 cycles of amplification. Samples were run on anagarose gel, and PCR products were quantitated using the AlphaImagersoftware. Expression was recorded as strong or medium, if signal isdetected as 26 or 30 cycles of amplification respectively, and absent ifno signal is detected even at 30 cycles of amplification. Results showexpression of STEAP-1 in 8 of 11 (72.7%) tumor specimens tested.

FIGS. 15A-15C. Specific cell Surface staining of STEAP-1 by MAbM2/92.30. Left panels: FACS analysis of recombinant 3T3 (FIG. 15A) andRat1 cells (FIG. 15B) stably expressing either STEAP1 (dark lines) or acontrol neomycin resistance gene (light lines) stained with anti-STEAPMAb M2/92.30 (10 μ/ml) and cell surface bound MAb was detected with agoat anti-mouse IgG-PE conjugate secondary reagent. The stained cellswere then subjected to FACS analysis. As indicated by the fluorescentshift of the Rat1-STEAP1 and 3T3-STEAP1 cells compared to the respectivecontrol cells, MAb M2/92.30 specifically binds to cell surface STEAP1.Right panel: Fluorescent microscopy of 3T3-STEAP1 cells stained with MAbM2/92.30 showing bright cell surface fluorescence (FIG. 15C).

FIG. 16. STEAP1 M2/92.30 MAb Recognizes Cell-Surface STEAP-1 on HumanProstate Cancer Xenografts. LAPC9 prostate cancer cells were stainedwith 10 μg/ml of either MAb M2/92.30 or with a control anti-KLH MAb.Surface bound MAb was detected with goat-anti-mouse IgG-PE conjugatedsecondary Ab. Stained cells were then subjected to FACS analysis. Theseresults demonstrate that the anti-STEAP1 MAb M2/120.545 specificallybinds endogenous cell surface STEAP1 expressed in prostate cancerxenograft cells.

FIG. 17. STEAP1 M2/92.30 MAb Recognizes Mouse STEAP-1. 293T cells weretransiently transfected with either pCDNA3.1 encoding the murine STEAP1cDNA or with an empty vector. 2 days later, the cells were harvested andstained with anti-STEAP1 MAb M2/92.30 (10 μg/ml) and cell surface boundMAb was detected with a goat anti-mouse IgG-PE conjugate secondaryreagent. Cells were then subjected to FACS analysis. As indicated by thefluorescent shift of the 293T cells transfected with murine STEAP1compared to the cells transfected with the empty vector, MAb M2/92.30specifically binds to murine STEAP1 protein.

FIGS. 18A-18D. STEAP1/120.545 MAb recognizes cell surface STEAP-1. FIGS.18A & 18B. 3T3-neo (FIG. 18A, filled histograms) and 3T3-STEAP1 cells(FIG. 18A, no fill histograms) and Rat1-neo (FIG. 18B, filledhistograms) and Rat1-STEAP cells (FIG. 18B, no fill histograms) werestained with MAb M2/120.545 (10 μg/ml) and surface bound MAb wasdetected with goat anti-mouse IgG-PE conjugated secondary Ab. Cells werethen subjected to FACS analysis. As indicated by the fluorescence shiftof the 3T3-STEAP1 and Rat1-STEAP1 cells compared to their respective neocontrols, MAb M2/120.545 specifically binds cell surface STEAP1. FIG.18C. LNCaP cells were stained with either MAb M2/120.545 or a controlanti-KLH MAb and subjected to FACS analysis as above. FIG. 18D.Fluorescence microscopy of the M2/120.545 stained LNCaP cells showingbright cell surface fluorescence. These results demonstrate that theM2/120.545 MAb specifically binds endogenous cell surface STEAP1 inLNCaP cells.

FIGS. 19A-19D. FIG. 19A The cDNA (SEQ ID NO: 49) and amino acid sequence(SEQ ID NO: 50) of M2/X92.30 VH clone #2. FIG. 19B The cDNA (SEQ ID NO:51) and amino acid sequence (SEQ ID NO: 52) of M2/X92.30 VL clone #2.FIG. 19C The cDNA (SEQ ID NO: 53) and amino acid sequence (SEQ ID NO:54) of M2/X92.30 VL clone #6. FIG. 19D The cDNA (SEQ ID NO: 55) andamino acid sequence (SEQ ID NO: 56) of M2/X120.545 VL clone #8.

FIGS. 20A-20E. FIG. 20A. The amino acid sequence (SEQ ID NO: 57) ofM2/X92.30 VH clone #2. FIG. 20B. The amino acid sequence (SEQ ID NO: 58)of M21X92.30 VL clone #2. FIG. 20C. The cDNA (SEQ ID NO: 59) and aminoacid sequence (SEQ ID NO: 60) of M2/X92.30 VL clone #6. FIG. 20D. Aminoacid alignment of M2/X92.30 VL clone #2 (SEQ ID NO: 61) and M2/M92.30 VLclone #6 (SEQ ID NO: 62). FIG. 20E. The amino acid sequence (SEQ ID NO:63) of M2/X120.545 VL clone #8. The sequence of the signal peptide isunderlined.

FIGS. 21A & 21B. STEAP1 M2/92.30 MAb Recognizes Cell-Surface STEAP-1 onHuman Prostate and Bladder Cancer Xenografts. UGB1 bladder cancer cells(FIG. 21A) and LAPC9 prostate cancer cells (FIG. 21B) were stained with10 μg/ml of either MAb M2/92.30 or with a control anti-KLH MAb. Surfacebound MAb was detected with goat-anti-mouse IgG-PE conjugated secondaryAb. Stained cells were then subjected to FACS analysis. These resultsdemonstrate that the anti-STEAP1 MAb M2/92.30 specifically bindsendogenous cell surface STEAP1 expressed in bladder and prostate cancerxenograft cells.

FIGS. 22A & 22B. STEAP-1 internalization by STEAP1/92.30 MAb. 3T3-STEAP1cells were stained at 4 C with M2/92.30 MAb (10 μg/ml), washed, thenincubated with goat anti-mouse IgG-PE conjugate secondary Ab at 4 C.One-half of the cells were moved to 37 C for 30 minutes and the otherhalf remained at 4 C. Cells from each treatment were then subjected tofluorescent microscopy. Cells that remained at 4 C showed bright“ring-like” cell surface fluorescence. Cells that were moved to 37 Cshowed loss of the “ring-like” cell surface fluorescence and theappearance of punctate and aggregated fluorescence indicative of cappingand internalization.

FIGS. 23A & 23B. STEAP-1 internalization by STEAP1 M2/120.545 MAb.PC3-STEAP1 cells were stained at 4 C with M2/120.545 MAb (10 μg/ml),washed, then incubated with goat anti-mouse IgG-PE conjugate secondaryAb. One-half of the cells were moved to 37 C for 30 minutes and theother half remained at 4 C. Cells from each treatment were thensubjected to fluorescent microscopy. Cells that remained at 4 C showedbright “ring-like” cell surface fluorescence. Cells that were moved to37 C showed loss of the “ring-like” cell surface fluorescence and theappearance of punctate and aggregated fluorescence indicative of cappingand internalization.

FIG. 24. STEAP-1 Internalization. Anti-mouse IgG—saporin conjugates(Advanced Targeting Systems, San Diego, Calif.) was used to demonstratethat murine Steap-1 M2/120.545 enters target cells via expression ofSteap-1 on the surface of LNCaP cells. The following protocols wereused. LNCaP cells were plated at 5000 cells/90 μl/well in 96-well plateand incubated overnight. Secondary immunotoxin conjugates (anti-mouseIgG-saporin and anti-goat IgG-saporin) or anti-mouse IgG were made incell culture medium to yield a final concentration of 100 ng/ml. Theprimary antibody was added at concentrations ranging from 1-1000 ng/ml.The plates were incubated for 72 hours and the viability was determinedby MTT assay. The results show that LNCaP cells were killed in thepresence of M2/120.545 and anti-mouse IgG-saporin. No effects weredetected with either the secondary antibody alone (anti-mouse IgG) ornonspecific secondary antibody conjugates (anti-goat IgG saporin). Notoxicity was observed with the primary antibody (M2/120.545) alonetested up to 1 μg/ml.

FIG. 25. Immunoprecipitation of STEAP1 by anti-STEAP-1 MAbs M2/92.30 andM2/120.545. 3T3-STEAP1 and 3T3-neo cells were lysed in RIPA buffer (25mM Tris-CI pH7.4; 150 mM NaCl, 0.5 mM EDTA, 1% Triton X-100, 0.5%deoxycholic acid, 0.1% SDS, and protease inhibitor cocktail). The celllysates were precleared with protein G sepharose beads and thenincubated with 5 ug of either MAb M2/92.30 or M2/120.545 for 2 hours atroom temperature. Protein G beads were added and the mixture was furtherincubated for 1 hour. The immune complexes were washed and solubilizedin SDS-PAGE sample buffer. The solubilized samples were then subjectedto SDS-PAGE and Western blot analysis using a rabbit anti-STEAP pAb.Whole cell lysates of 293T cells transfected with STEAP1 was also run asa positive control. An immunoreactive band of ˜37 kD was seen only insamples derived from 3T3-STEAP1 cells indicative of specificimmunoprecipitation of STEAP1 by both M2/92.30 and M2/120.545 MAbs.

FIGS. 26A & 26B. Effect of STEAP-1 MAbs on the Growth of LAPC9 HumanProstate Cancer Xenografts in Mice. STEAP-1 M2/92.30 and M2/120.545 weretested at two different doses of 100 μg and 500 μg. PBS and anti-KLH MAbwere used as controls. The study cohort consisted of 6 groups with 10mice in each group. MAbs were dosed IP twice a week for a total of 12doses, starting the same day as tumor cell injection. Tumor size wasmonitored through caliper measurements twice a week. The longestdimension (L) and the dimension perpendicular to it (W) were taken tocalculate tumor volume using the formula: W²×L/2. Serum PSAconcentration at treatment day 40 for each animal was measured usingcommercial ELISA kit. The Kruskal-Wallis test and the Mann-Whitney Utest were used to evaluate differences of tumor growth and PSA levelamong groups. All tests were two-sided with {dot over (a)}=0.05. Thedata show that STEAP-1 M2/92.30 and M2/120.545 significantly retard thegrowth of human prostate xenograft in a dose-dependent tumor.

FIG. 27. Effect of STEAP-1 MAbs on the Growth of LAPC9 Human ProstateCancer Xenograft in Mice. STEAP-1 M2/92.30 and M2/120.545 were tested attwo different doses of 100 μg and 500 μg. PBS and anti-KLH MAb were usedas controls. The study cohort consisted of 6 groups with 10 mice in eachgroup. MAbs were dosed IP twice a week for a total of 12 doses, startingthe same day as tumor cell injection. Tumor size was monitored throughcaliper measurements twice a week. The longest dimension (L) and thedimension perpendicular to it (W) were taken to calculate tumor volumeusing the formula: W²×L/2. Serum PSA concentration at treatment day 40for each animal was measured using commercial ELISA kit. TheKruskal-Wallis test and the Mann-Whitney U test were used to evaluatedifferences of tumor growth and PSA level among groups. All tests weretwo-sided with {dot over (a)}=0.05. The results show that STEAP-1M2/92.30 and M2/120.545 significantly retard the growth of humanprostate xenograft in a dose-dependent tumor.

FIG. 28. STEAP-1 induced ERK-1 and ERK-2 phosphorylation. Left panels:PC3 cells were transfected with neomycin resistance gene alone or withSTEAP-1 in pSRa vector. Cells were grown overnight in 0.5% FBS, thenstimulated with 10% FBS for 5 minutes with or without 10 μg/ml MEKinhibitor PD98058. Cell lysates were resolved by 12.5% SDS-PAGE andWestern blotted with anti-phospho-ERK (Cell Signaling) andanti-ERK(Zymed). Right panels: NIH-3T3 cells were transfected withneomycin resistance gene alone or with STEAP-1 in pSRa vector. Cellswere treated as above but without the MEK inhibitor. In addition,NIH-3T3-Neo cells were treated with 10 mg/ml Na salycilate. Expressionof STEAP-1 induces the phosphorylation of ERK-1 and ERK-2 in serum andwas inhibited by the upstream MEK kinase inhibitor PD98058.

FIG. 29. STEAP-1 Mediates Cell-Cell Communication. PC3 cells weretransfected with neomycin resistance gene alone or with STEAP-1 or acontrol gene in pSRa vector. Recipient cells were labeled with 1 mg/mldextran-Texas Red and donor cells were labeled with 2.5 μg/ml calceinAM. The donor (green) and recipient (red) cells were co-cultured at 37°C. for 18-24 hours and analyzed by microscopy for the co-localization offluorescent dyes. Left panel: PC3-Neo cells were used as both donor andrecipient. Center panel: PC3-STEAP-1 cells were used as both donor andrecipient. Right panel: PC3-control cells were used as both donor andrecipient. STEAP-1 induced the transfer of calcein to cells containingdextran-Texas Red, indicating that STEAP-1 facilitates cell-cellcommunication.

FIG. 30. Cell Communication Requires STEAP-1 Expression on Donor andRecipient Cells. PC3 cells were transfected with neomycin resistancegene alone or with STEAP-1 in pSRa vector. Recipient cells were labeledwith 1 mg/ml dextran-Texas Red and donor cells were labeled with 2.5μg/ml calcein AM. The donor (green) and recipient (red) cells wereco-cultured at 37° C. for 18-24 hours and analyzed by microscopy for theco-localization of fluorescent dyes. Upper panels: light microscopy;lower panels: UV fluorescence. Left panels: PC3-Neo cells were bothdonor and recipient. Center panels: PC3-Neo were donor cells andPC3-STEAP-1 were recipient. Right panels: PC3-STEAP-1 cells were bothdonor and recipient. Only when STEAP-1 was expressed on both donor andrecipient was cell-cell communication detected.

FIG. 31. STEAP-1/120.545 MAb Effect on Gap Junction. PC3 cells weretransfected with neomycin resistance gene alone or with STEAP-1 in pSRavector. Recipient cells were labeled with 1 mg/ml dextran-Texas Red anddonor cells were labeled with 2.5 μg/ml calcein AM. The donor (green)and recipient (red) cells were co-cultured at 37° C. for 18-24 hours andanalyzed by microscopy for the co-localization of fluorescent dyes. Inall experiments, the same cells were used as donor and acceptor. Cellswere incubated with the indicated amounts of STEAP-1/120.545 MAb for 10minutes prior to plating and MAb was maintained in the culture for 24hours prior to analysis. STEAP1/120.545 reduces STEAP-1 mediated gapjunction in a dose-dependent manner.

FIG. 32. Inhibition of ERK-1 and ERK-2 phosphorylation by STEAP-1 MAband RNAi. PC3 cells were transfected with neomycin resistance gene aloneor with STEAP-1 and MAb in pSRa vector. For RNAi knockdown, PC3-STEAP-1cells were stably transfected with a pPUR-U6-27-STEAP-1 vectorcontaining siRNA to STEAP-1. Cells were starved in 0.1% FBS for 18 hoursat 37° C., placed on ice for 10 minutes without or with 10 μg/mlM2/92.30 MAb, brought to RT for 3 minutes then stimulated with 10% FBSfor 5 minutes. Cells were lysed in RIPA buffer, whole cell lysatesresolved by 12.5% SDS-PAGE and proteins detected by Western blotting.Phospho-ERK was detected with rabbit antiserum (Cell Signaling) and ERKwas detected with rabbit anti-ERK (Zymed). STEAP-1 was detected withsheep anti-STEAP-1 and actin was detected with anti-actin MAb (SantaCruz). ERK-1 and ERK-2 phosphorylation were both induced by 10% serum,and were inhibited by M2/92.30 MAb and siRNA to STEAP-1.

FIG. 33. Effect of STEAP-1 RNAi on Cell-Cell Communication. PC3 cellswere transfected with neomycin resistance gene alone or with STEAP-1 inpSRa vector. For RNAi knockdown, PC3-STEAP-1 cells were stablytransfected with a pPUR-U6-27-STEAP-1 vector containing siRNA to STEAP-1or an empty vector. Recipient cells were labeled with 1 mg/mldextran-Texas Red and donor cells were labeled with 2.5 μg/ml calceinAM. The donor (green) and recipient (red) cells were co-cultured at 37°C. for 18-24 hours and analyzed by microscopy for the co-localization offluorescent dyes. In all experiments, the same cells were used as donorand acceptor. Specific STEAP-1 RNAi stably expressed in PC3-STEAP-1cells reduces the STEAP-1 induced cell-cell communication.

DETAILED DESCRIPTION OF THE INVENTION

Outline of Sections

I.) Definitions

II.) STEAP-1 Polynucleotides

II.A.) Uses of STEAP-1 Polynucleotides

II.A.1.) Monitoring of Genetic Abnormalities

IIA.2.) Antisense Embodiments

IIA.3.) Primers and Primer Pairs

-   -   II.A.4.) Isolation of STEAP-1-Encoding Nucleic Acid Molecules

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

III.) STEAP-1-related Proteins

-   -   III.A.) Motif-bearing Protein Embodiments    -   III.B.) Expression of STEAP-1-related Proteins    -   III.C.) Modifications of STEAP-1-related Proteins    -   III.D.) Uses of STEAP-1-related Proteins

IV.) STEAP-1 Antibodies

V.) STEAP-1 Cellular Immune Responses

VI.) STEAP-1 Transgenic Animals

VII.) Methods for the Detection of STEAP-1

VIII.) Methods for Monitoring the Status of STEAP-1-related Genes andTheir Products

IX.) Identification of Molecules That Interact With STEAP-1

X.) Therapeutic Methods and Compositions

-   -   X.A.) Anti-Cancer Vaccines

X.B.) STEAP-1 as a Target for Antibody-Based Therapy

X.C.) STEAP-1 as a Target for Cellular Immune Responses

-   -   X.C.1. Minigene Vaccines    -   X.C.2. Combinations of CTL Peptides with Helper Peptides    -   X.C.3. Combinations of CTL Peptides with T Cell Priming Agents    -   X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or        HTL Peptides    -   X.D.) Adoptive Immunotherapy

X.E.) Administration of Vaccines for Therapeutic or ProphylacticPurposes

XI.) Diagnostic and Prognostic Embodiments of STEAP-1.

XII.) Inhibition of STEAP-1 Protein Function

-   -   XII.A.) Inhibition of STEAP-1 With Intracellular Antibodies    -   XII.B.) Inhibition of STEAP-1 with Recombinant Proteins    -   XII.C.) Inhibition of STEAP-1 Transcription or Translation    -   XII.D.) General Considerations for Therapeutic Strategies

XIII.) Identification, Characterization and Use of Modulators of STEAP-1

XIV.) RNAi and Therapeutic use of small interfering RNA (siRNAs)

XV.) KITS/Articles of Manufacture

I.) DEFINITIONS

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al, Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostatecancer”, “advanced disease” and “locally advanced disease” mean prostatecancers that have extended through the prostate capsule, and are meantto include stage C disease under the American Urological Association(AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, andstage T3-T4 and N+ disease under the TNM (tumor, node, metastasis)system. In general, surgery is not recommended for patients with locallyadvanced disease, and these patients have substantially less favorableoutcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence STEAP-1 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence STEAP-1. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g. aSTEAP-1-related protein). For example, an analog of a STEAP-1 proteincan be specifically bound by an antibody or T cell that specificallybinds to STEAP-1.

The term “antibody” is used in the broadest sense unless clearlyindicated otherwise. Therefore, an “antibody” can be naturally occurringor man-made such as monoclonal antibodies produced by conventionalhybridoma technology. Anti-STEAP-1 antibodies comprise monoclonal andpolyclonal antibodies as well as fragments containing theantigen-binding domain and/or one or more complementarily determiningregions of these antibodies. As used herein, the term “antibody” refersto any form of antibody or fragment thereof that specifically bindsSTEAP-1 and/or exhibits the desired biological activity and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as theyspecifically bind STEAP-1 and/or exhibit the desired biologicalactivity. Any specific antibody can be used in the methods andcompositions provided herein. Thus, in one embodiment the term“antibody” encompasses a molecule comprising at least one variableregion from a light chain immunoglobulin molecule and at least onevariable region from a heavy chain molecule that in combination form aspecific binding site for the target antigen. In one embodiment, theantibody is an IgG antibody. For example, the antibody is a IgG₁, IgG₂,IgG3, or IgG4 antibody. The antibodies useful in the present methods andcompositions can be generated in cell culture, in phage, or in variousanimals, including but not limited to cows, rabbits, goats, mice, rats,hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes.Therefore, in one embodiment, an antibody of the present invention is amammalian antibody. Phage techniques can be used to isolate an initialantibody or to generate variants with altered specificity or aviditycharacteristics. Such techniques are routine and well known in the art.In one embodiment, the antibody is produced by recombinant means knownin the art. For example, a recombinant antibody can be produced bytransfecting a host cell with a vector comprising a DNA sequenceencoding the antibody. One or more vectors can be used to transfect theDNA sequence expressing at least one VL and one VH region in the hostcell. Exemplary descriptions of recombinant means of antibody generationand production include Delves, ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES(Wiley, 1997); Shephard, et al., MONOCLONAL ANTIBODIES (OxfordUniversity Press, 2000); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (Academic Press, 1993); CURRENT PROTOCOLS IN IMMUNOLOGY (JohnWiley & Sons, most recent edition). An antibody of the present inventioncan be modified by recombinant means to increase greater efficacy of theantibody in mediating the desired function. Thus, it is within the scopeof the invention that antibodies can be modified by substitutions usingrecombinant means. Typically, the substitutions will be conservativesubstitutions. For example, at least one amino acid in the constantregion of the antibody can be replaced with a different residue. See,e.g., U.S. Pat. No. 5,624,821, U.S. Pat. No. 6,194,551, Application No.WO 9958572; and Angel, et al., Mol. Immunol 30:105-08 (1993). Themodification in amino acids includes deletions, additions, substitutionsof amino acids. In some cases, such changes are made to reduce undesiredactivities, e.g., complement-dependent cytotoxicity. Frequently, theantibodies are labeled by joining, either covalently or non-covalently,a substance which provides for a detectable signal. A wide variety oflabels and conjugation techniques are known and are reported extensivelyin both the scientific and patent literature. These antibodies can bescreened for binding to normal or defective STEAP-1. See e.g., ANTIBODYENGINEERING: A PRACTICAL APPROACH (Oxford University Press, 1996).Suitable antibodies with the desired biologic activities can beidentified the following in vitro assays including but not limited to:proliferation, migration, adhesion, soft agar growth, angiogenesis,cell-cell communication, apoptosis, transport, signal transduction, andthe following in vivo assays such as the inhibition of tumor growth. Theantibodies provided herein can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bescreened for the ability to bind to the specific antigen withoutinhibiting the receptor-binding or biological activity of the antigen.As neutralizing antibodies, the antibodies can be useful in competitivebinding assays. They can also be used to quantify the STEAP-1 or itsreceptor.

An “antibody fragment” is defined as at least a portion of the variableregion of the immunoglobulin molecule that binds to its target, i.e.,the antigen-binding region. In one embodiment it specifically coverssingle anti-STEAP-1 antibodies and clones thereof (including agonist,antagonist and neutralizing antibodies) and anti-STEAP-1 antibodycompositions with polyepitopic specificity. The antibody of the presentmethods and compositions can be monoclonal or polyclonal. An antibodycan be in the form of an antigen binding antibody fragment including aFab fragment, F(ab′)₂ fragment, a single chain variable region, and thelike. Fragments of intact molecules can be generated using methods wellknown in the art and include enzymatic digestion and recombinant means.

As used herein, any form of the “antigen” can be used to generate anantibody that is specific for STEAP-1. Thus, the eliciting antigen maybe a single epitope, multiple epitopes, or the entire protein alone orin combination with one or more immunogenicity enhancing agents known inthe art. The eliciting antigen may be an isolated full-length protein, acell surface protein (e.g., immunizing with cells transfected with atleast a portion of the antigen), or a soluble protein (e.g., immunizingwith only the extracellular domain portion of the protein). The antigenmay be produced in a genetically modified cell. The DNA encoding theantigen may genomic or non-genomic (e.g., cDNA) and encodes at least aportion of the extracellular domain. As used herein, the term “portion”refers to the minimal number of amino acids or nucleic acids, asappropriate, to constitute an immunogenic epitope of the antigen ofinterest. Any genetic vectors suitable for transformation of the cellsof interest may be employed, including but not limited to adenoviralvectors, plasmids, and non-viral vectors, such as cationic lipids. Inone embodiment, the antibody of the methods and compositions hereinspecifically bind at least a portion of the extracellular domain of theSTEAP-1 of interest.

The antibodies or antigen binding fragments thereof provided herein maybe conjugated to a “bioactive agent.” As used herein, the term“bioactive agent” refers to any synthetic or naturally occurringcompound that binds the antigen and/or enhances or mediates a desiredbiological effect to enhance cell-killing toxins.

In one embodiment, the binding fragments useful in the present inventionare biologically active fragments. As used herein, the term“biologically active” refers to an antibody or antibody fragment that iscapable of binding the desired the antigenic epitope and directly orindirectly exerting a biologic effect. Direct effects include, but arenot limited to the modulation, stimulation, and/or inhibition of agrowth signal, the modulation, stimulation, and/or inhibition of ananti-apoptotic signal, the modulation, stimulation, and/or inhibition ofan apoptotic or necrotic signal, modulation, stimulation, and/orinhibition the ADCC cascade, and modulation, stimulation, and/orinhibition the CDC cascade.

“Bispecific” antibodies are also useful in the present methods andcompositions. As used herein, the term “bispecific antibody” refers toan antibody, typically a monoclonal antibody, having bindingspecificities for at least two different antigenic epitopes. In oneembodiment, the epitopes are from the same antigen. In anotherembodiment, the epitopes are from two different antigens. Methods formaking bispecific antibodies are known in the art. For example,bispecific antibodies can be produced recombinantly using theco-expression of two immunoglobulin heavy chain/light chain pairs. See,e.g., Milstein et al., Nature 305:537-39 (1983). Alternatively,bispecific antibodies can be prepared using chemical linkage. See, e.g.,Brennan, et al., Science 229:81 (1985). Bispecific antibodies includebispecific antibody fragments. See, e.g., Hollinger, et al., Proc. Natl.Acad. Sci. U.S.A. 90:6444-48 (1993), Gruber, et al., J. Immunol.152:5368 (1994).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they specifically bindthe target antigen and/or exhibit the desired biological activity (U.S.Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The term “codon optimized sequences” refers to nucleotide sequences thathave been optimized for a particular host species by replacing anycodons having a usage frequency of less than about 20%. Nucleotidesequences that have been optimized for expression in a given hostspecies by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

A “combinatorial library” is a collection of diverse chemical compoundsgenerated by either chemical synthesis or biological synthesis bycombining a number of chemical “building blocks” such as reagents. Forexample, a linear combinatorial chemical library, such as a polypeptide(e.g., mutein) library, is formed by combining a set of chemicalbuilding blocks called amino acids in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptidecompound). Numerous chemical compounds are synthesized through suchcombinatorial mixing of chemical building blocks (Gallop et al., J. Med.Chem. 37(9): 1233-1251 (1994)).

Preparation and screening of combinatorial libraries is well known tothose of skill in the art. Such combinatorial chemical librariesinclude, but are not limited to, peptide libraries (see, e.g., U.S. Pat.No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493 (1991), Houghton etal., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO91/19735), encoded peptides (PCT Publication WO 93/20242), randombio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat.No. 5,288,514), diversomers such as hydantoins, benzodiazepines anddipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913(1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.114:6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucosescaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218(1992)), analogous organic syntheses of small compound libraries (Chenet al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho, etal., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell etal., J. Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J.Med. Chem. 37:1385 (1994), nucleic acid libraries (see, e.g.,Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat.No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., NatureBiotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydratelibraries (see, e.g., Liang et al., Science 274:1520-1522 (1996), andU.S. Pat. No. 5,593,853), and small organic molecule libraries (see,e.g., benzodiazepines, Baum, C&EN, January 18, page 33 (1993);isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, LouisvilleKy.; Symphony, Rainin, Woburn, Mass.; 433A, Applied Biosystems, FosterCity, Calif.; 9050, Plus, Millipore, Bedford, NIA). A number ofwell-known robotic systems have also been developed for solution phasechemistries. These systems include automated workstations such as theautomated synthesis apparatus developed by Takeda Chemical Industries,LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms(Zymate H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard,Palo Alto, Calif.), which mimic the manual synthetic operationsperformed by a chemist. Any of the above devices are suitable for usewith the present invention. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex,Moscow, RU; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, RU; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.; etc.).

As used herein, the term “conservative substitution” refers tosubstitutions of amino acids are known to those of skill in this art andmay be made generally without altering the biological activity of theresulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson, et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin/CummingsPub. Co., p. 224 (4th Edition 1987)). Such exemplary substitutions arepreferably made in accordance with those set forth in Table(s) III(a-b).For example, such changes include substituting any of isoleucine (I),valine (V), and leucine (L) for any other of these hydrophobic aminoacids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine(Q) for asparagine (N) and vice versa; and serine (S) for threonine (T)and vice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments (see, e.g. Table III(a)herein; pages 13-15 “Biochemistry” 2^(nd) ED. Lubert Stryer ed (StanfordUniversity); Henikoff et al, PNAS 1992 Vol 89 10915-10919; Lei et al., JBiol Chem 1995 May 19; 270(20):11882-6). Other substitutions are alsopermissible and may be determined empirically or in accord with knownconservative substitutions.

The term “cytotoxic agent” refers to a substance that inhibits orprevents the expression activity of cells, function of cells and/orcauses destruction of cells. The term is intended to include radioactiveisotopes chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to auristatins, auristatine, auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain,combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin,taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracindione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40,abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin,mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin,calicheamicin, Sapaonana officinalis inhibitor, and glucocorticoid andother chemotherapeutic agents, as well as radioisotopes such as At²¹¹,I¹³¹, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, SM¹⁵³, Bi²¹², or ²¹³, P³², and radioactiveisotopes of Lu including Lu¹⁷⁷. Antibodies may also be conjugated to ananti-cancer pro-drug activating enzyme capable of converting thepro-drug to its active form.

As used herein, the term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L).). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementary domains ofanother chain and create two antigen-binding sites. Diabodies aredescribed more fully in, e.g., EP 404,097; WO 93/11161; and Hollinger etal., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993).

The “gene product” is used herein to indicate a peptide/protein or mRNA.For example, a “gene product of the invention” is sometimes referred toherein as a “cancer amino acid sequence”, “cancer protein”, “protein ofa cancer listed in Table I”, a “cancer mRNA”, “mRNA of a cancer listedin Table I”, etc. In one embodiment, the cancer protein is encoded by anucleic acid of FIG. 2. The cancer protein can be a fragment, oralternatively, be the full-length protein encoded by nucleic acids ofFIG. 2. In one embodiment, a cancer amino acid sequence is used todetermine sequence identity or similarity. In another embodiment, thesequences are naturally occurring allelic variants of a protein encodedby a nucleic acid of FIG. 2. In another embodiment, the sequences aresequence variants as further described herein.

“Heteroconjugate” antibodies are useful in the present methods andcompositions. As used herein, the term “heteroconjugate antibody” refersto two covalently joined antibodies. Such antibodies can be preparedusing known methods in synthetic protein chemistry, including usingcrosslinking agents. See, e.g., U.S. Pat. No. 4,676,980.

“High throughput screening” assays for the presence, absence,quantification, or other properties of particular nucleic acids orprotein products are well known to those of skill in the art. Similarly,binding assays and reporter gene assays are similarly well known. Thus,e.g., U.S. Pat. No. 5,559,410 discloses high throughput screeningmethods for proteins; U.S. Pat. No. 5,585,639 discloses high throughputscreening methods for nucleic acid binding (i.e., in arrays); while U.S.Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methods ofscreening for ligand/antibody binding.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Amersham Biosciences, Piscataway, N.J.; ZymartCorp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; BeckmanInstruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick,Mass.; etc.). These systems typically automate entire procedures,including all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthroughput and rapid start up as well as a high degree of flexibilityand customization. The manufacturers of such systems provide detailedprotocols for various high throughput systems. Thus, e.g., Zymark Corp.provides technical bulletins describing screening systems for detectingthe modulation of gene transcription, ligand binding, and the like.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

In one embodiment, the antibody provided herein is a “human antibody.”As used herein, the term “human antibody” refers to an antibody in whichessentially the entire sequences of the light chain and heavy chainsequences, including the complementary determining regions (CDRs), arefrom human genes. In one embodiment, human monoclonal antibodies areprepared by the trioma technique, the human B-cell technique (see, e.g.,Kozbor, et al., Immunol. Today 4: 72 (1983), EBV transformationtechnique (see, e.g., Cole et al. MONOCLONAL ANTIBODIES AND CANCERTHERAPY 77-96 (1985)), or using phage display (see, e.g., Marks et al.,J. Mol. Biol. 222:581 (1991)). In a specific embodiment, the humanantibody is generated in a transgenic mouse. Techniques for making suchpartially to fully human antibodies are known in the art and any suchtechniques can be used. According to one particularly preferredembodiment, fully human antibody sequences are made in a transgenicmouse engineered to express human heavy and light chain antibody genes.An exemplary description of preparing transgenic mice that produce humanantibodies found in Application No. WO 02/43478 and U.S. Pat. No.6,657,103 (Abgenix) and its progeny. B cells from transgenic mice thatproduce the desired antibody can then be fused to make hybridoma celllines for continuous production of the antibody. See, e.g., U.S. Pat.Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; andJakobovits, Adv. Drug Del. Rev. 31:33-42 (1998); Green, et al., J. Exp.Med. 188:483-95 (1998).

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., murine)antibodies as well as human antibodies. Such antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. See e.g., Cabilly U.S. Pat. No. 4,816,567; Queen et al.(1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; and ANTIBODYENGINEERING: A PRACTICAL APPROACH (Oxford University Press 1996).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used inthe context of polynucleotides, are meant to refer to conventionalhybridization conditions, preferably such as hybridization in 50%formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures forhybridization are above 37 degrees C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55 degrees C.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment. For example, a polynucleotide is said to be “isolated” whenit is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the STEAP-1 genes orthat encode polypeptides other than STEAP-1 gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated STEAP-1 polynucleotide. A protein issaid to be “isolated,” for example, when physical, mechanical orchemical methods are employed to remove the STEAP-1 proteins fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated STEAP-1 protein. Alternatively, an isolated proteincan be prepared by chemical means.

Suitable “labels” include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. In addition, the antibodies provided hereincan be useful as the antigen-binding component of fluorobodies. Seee.g., Zeytun et al., Nat. Biotechnol. 21:1473-79 (2003).

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” meanprostate cancers that have spread to regional lymph nodes or to distantsites, and are meant to include stage D disease under the AUA system andstage T×N×M+ under the TNM system. As is the case with locally advancedprostate cancer, surgery is generally not indicated for patients withmetastatic disease, and hormonal (androgen ablation) therapy is apreferred treatment modality. Patients with metastatic prostate cancereventually develop an androgen-refractory state within 12 to 18 monthsof treatment initiation. Approximately half of these androgen-refractorypatients die within 6 months after developing that status. The mostcommon site for prostate cancer metastasis is bone. Prostate cancer bonemetastases are often osteoblastic rather than osteolytic (i.e.,resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

The term “modulator” or “test compound” or “drug candidate” orgrammatical equivalents as used herein describe any molecule, e.g.,protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., to be tested for the capacity to directly orindirectly alter the cancer phenotype or the expression of a cancersequence, e.g., a nucleic acid or protein sequences, or effects ofcancer sequences (e.g., signaling, gene expression, protein interaction,etc.) In one aspect, a modulator will neutralize the effect of a cancerprotein of the invention. By “neutralize” is meant that an activity of aprotein is inhibited or blocked, along with the consequent effect on thecell. In another aspect, a modulator will neutralize the effect of agene, and its corresponding protein, of the invention by normalizinglevels of said protein. In preferred embodiments, modulators alterexpression profiles, or expression profile nucleic acids or proteinsprovided herein, or downstream effector pathways. In one embodiment, themodulator suppresses a cancer phenotype, e.g. to a normal tissuefingerprint. In another embodiment, a modulator induced a cancerphenotype. Generally, a plurality of assay mixtures is run in parallelwith different agent concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e., at zero concentration or below thelevel of detection.

Modulators, drug candidates or test compounds encompass numerouschemical classes, though typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 100 and less than about 2,500 Daltons. Preferred small moleculesare less than 2000, or less than 1500 or less than 1000 or less than 500D. Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Modulators also comprise biomolecules such aspeptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof. Particularlypreferred are peptides. One class of modulators are peptides, forexample of from about five to about 35 amino acids, with from about fiveto about 20 amino acids being preferred, and from about 7 to about 15being particularly preferred. Preferably, the cancer modulatory proteinis soluble, includes a non-transmembrane region, and/or, has anN-terminal Cys to aid in solubility. In one embodiment, the C-terminusof the fragment is kept as a free acid and the N-terminus is a freeamine to aid in coupling, i.e., to cysteine. In one embodiment, a cancerprotein of the invention is conjugated to an immunogenic agent asdiscussed herein. In one embodiment, the cancer protein is conjugated toBSA. The peptides of the invention, e.g., of preferred lengths, can belinked to each other or to other amino acids to create a longerpeptide/protein. The modulatory peptides can be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. In a preferred embodiment, peptide/protein-basedmodulators are antibodies, and fragments thereof, as defined herein.

Modulators of cancer can also be nucleic acids. Nucleic acid modulatingagents can be naturally occurring nucleic acids, random nucleic acids,or “biased” random nucleic acids. For example, digests of prokaryotic oreukaryotic genomes can be used in an approach analogous to that outlinedabove for proteins.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. In contrast, conventional(polyclonal) antibody preparations typically include a multitude ofantibodies directed against (or specific for) different epitopes. In oneembodiment, the polyclonal antibody contains a plurality of monoclonalantibodies with different epitope specificities, affinities, oravidities within a single antigen that contains multiple antigenicepitopes. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256: 495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352: 624-628 (1991) and Marks et al., J. MolBiol. 222: 581-597 (1991), for example. These monoclonal antibodies willusually bind with at least a K_(d) of about 1 nM, more usually at leastabout 300 nM, typically at least about 30 nM, preferably at least about10 nM, more preferably at least about 3 nM or better, usually determinedby ELISA.

A “motif”, as in biological motif of a STEAP-1-related protein, refersto any pattern of amino acids forming part of the primary sequence of aprotein, that is associated with a particular function (e.g.protein-protein interaction, protein-DNA interaction, etc) ormodification (e.g. that is phosphorylated, glycosylated or amidated), orlocalization (e.g. secretory sequence, nuclear localization sequence,etc.) or a sequence that is correlated with being immunogenic, eitherhumorally or cellularly. A motif can be either contiguous or capable ofbeing aligned to certain positions that are generally correlated with acertain function or property. In the context of HLA motifs, “motif”refers to the pattern of residues in a peptide of defined length,usually a peptide of from about 8 to about 13 amino acids for a class IHLA motif and from about 6 to about 25 amino acids for a class II HLAmotif, which is recognized by a particular HLA molecule. Peptide motifsfor HLA binding are typically different for each protein encoded by eachhuman HLA allele and differ in the pattern of the primary and secondaryanchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T), as shown for example in FIG. 2, can also beuracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific positionalong a peptide sequence which is understood to provide a contact pointbetween the immunogenic peptide and the HLA molecule. One to three,usually two, primary anchor residues within a peptide of defined lengthgenerally defines a “motif” for an immunogenic peptide. These residuesare understood to fit in close contact with peptide binding groove of anHLA molecule, with their side chains buried in specific pockets of thebinding groove. In one embodiment, for example, the primary anchorresidues for an HLA class I molecule are located at position 2 (from theamino terminal position) and at the carboxyl terminal position of a 8,9, 10, 11, or 12 residue peptide epitope in accordance with theinvention. Alternatively, in another embodiment, the primary anchorresidues of a peptide binds an HLA class II molecule are spaced relativeto each other, rather than to the termini of a peptide, where thepeptide is generally of at least 9 amino acids in length. The primaryanchor positions for each motif and supermotif are set forth in TableIV(a). For example, analog peptides can be created by altering thepresence or absence of particular residues in the primary and/orsecondary anchor positions shown in Table IV. Such analogs are used tomodulate the binding affinity and/or population coverage of a peptidecomprising a particular HLA motif or supermotif.

“Radioisotopes” include, but are not limited to the following(non-limiting exemplary uses are also set forth in Table IV(I)).

By “randomized” or grammatical equivalents as herein applied to nucleicacids and proteins is meant that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. Theserandom peptides (or nucleic acids, discussed herein) can incorporate anynucleotide or amino acid at any position. The synthetic process can bedesigned to generate randomized proteins or nucleic acids, to allow theformation of all or most of the possible combinations over the length ofthe sequence, thus forming a library of randomized candidate bioactiveproteinaceous agents.

In one embodiment, a library is “fully randomized,” with no sequencepreferences or constants at any position. In another embodiment, thelibrary is a “biased random” library. That is, some positions within thesequence either are held constant, or are selected from a limited numberof possibilities. For example, the nucleotides or amino acid residuesare randomized within a defined class, e.g., of hydrophobic amino acids,hydrophilic residues, sterically biased (either small or large)residues, towards the creation of nucleic acid binding domains, thecreation of cysteines, for cross-linking, prolines for SH-3 domains,serines, threonines, tyrosines or histidines for phosphorylation sites,etc., or to purines, etc.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that hasbeen subjected to molecular manipulation in vitro.

As used herein, the term “single-chain Fv” or “scFv” or “single chain”antibody refers to antibody fragments comprising the V_(H) and V_(L)domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

Non-limiting examples of “small molecules” include compounds that bindor interact with STEAP-1, ligands including hormones, neuropeptides,chemokines, odorants, phospholipids, and functional equivalents thereofthat bind and preferably inhibit STEAP-1 protein function. Suchnon-limiting small molecules preferably have a molecular weight of lessthan about 10 kDa, more preferably below about 9, about 8, about 7,about 6, about 5 or about 4 kDa. In certain embodiments, small moleculesphysically associate with, or bind, STEAP-1 protein; are not found innaturally occurring metabolic pathways; and/or are more soluble inaqueous than non-aqueous solutions.

As used herein, the term “specific” refers to the selective binding ofthe antibody to the target antigen epitope. Antibodies can be tested forspecificity of binding by comparing binding to appropriate antigen tobinding to irrelevant antigen or antigen mixture under a given set ofconditions. If the antibody binds to the appropriate antigen at least 2,5, 7, and preferably 10 times more than to irrelevant antigen or antigenmixture then it is considered to be specific. In one embodiment, aspecific antibody is one that only binds the STEAP-1 antigen, but doesnot bind to the irrelevant antigen. In another embodiment, a specificantibody is one that binds human STEAP-1 antigen but does not bind anon-human STEAP-1 antigen with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater amino acid homology with theSTEAP-1 antigen. In another embodiment, a specific antibody is one thatbinds human STEAP-1 antigen and binds murine STEAP-1 antigen, but with ahigher degree of binding the human antigen. In another embodiment, aspecific antibody is one that binds human STEAP-1 antigen and bindsprimate STEAP-1 antigen, but with a higher degree of binding the humanantigen. In another embodiment, the specific antibody binds to humanSTEAP-1 antigen and any non-human STEAP-1 antigen, but with a higherdegree of binding the human antigen or any combination thereof.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium. citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Press, 1989, and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 65° C. in asolution comprising: 1% bovine serum albumin, 0.5M sodium phosphatepH7.5, 1.25 mM EDTA, and 7% SDS 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), followed by washing the filters in 2×SSC/1% SDS at 50° C. and0.2×SSC/0.1% SDS at 50° C. The skilled artisan will recognize how toadjust the temperature, ionic strength, etc. as necessary to accommodatefactors such as probe length and the like.

An HLA “supennotif” is a peptide binding specificity shared by HLAmolecules encoded by two or more HLA alleles. Overall phenotypicfrequencies of HLA-supertypes in different ethnic populations are setforth in Table IV (f). The non-limiting constituents of varioussupertypes are as follows:

-   -   A2: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*6802,        A*6901, A*0207    -   A3: A3, A11, A31, A*3301, A*6801, A*0301, A*1101, A*3101    -   B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601,        B*6701, B*7801, B*0702, B*5101, B*5602    -   B44: B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)    -   A1: A*0102, A*2604, A*3601, A*4301, A*8001    -   A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003    -   B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02,        B*3901, B*3902, B*3903-04, B*4801-02, B*7301, B*2701-08    -   B58: B*1516, B*1517, B*5701, B*5702, B58    -   B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513 (B77)

Calculated population coverage afforded by different HLA-supertypecombinations are set forth in Table IV(g).

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; as isreadily appreciated in the art, full eradication of disease is apreferred out albeit not a requirement for a treatment act.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal, e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is acomposition that contains or encodes one or more peptides of theinvention. There are numerous embodiments of such vaccines, such as acocktail of one or more individual peptides; one or more peptides of theinvention comprised by a polyepitopic peptide; or nucleic acids thatencode such individual peptides or polypeptides, e.g., a minigene thatencodes a polyepitopic peptide. The “one or more peptides” can includeany whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides ofthe invention. The peptides or polypeptides can optionally be modified,such as by lipidation, addition of targeting or other sequences. HLAclass I peptides of the invention can be admixed with, or linked to, HLAclass II peptides, to facilitate activation of both cytotoxic Tlymphocytes and helper T lymphocytes. HLA vaccines can also comprisepeptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the STEAP-1 protein shown in FIG. 2 or FIG. 3.An analog is an example of a variant protein. Splice isoforms and singlenucleotides polymorphisms (SNPs) are further examples of variants.

The “STEAP-1-related proteins” of the invention include thosespecifically identified herein, as well as allelic variants,conservative substitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different STEAP-1 proteins orfragments thereof, as well as fusion proteins of a STEAP-1 protein and aheterologous polypeptide are also included. Such STEAP-1 proteins arecollectively referred to as the STEAP-1-related proteins, the proteinsof the invention, or STEAP-1. The term “STEAP-1-related protein” refersto a polypeptide fragment or a STEAP-1 protein sequence of 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, ormore than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65,70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275,300, 325, 330, 335, 339 or more amino acids.

II.) STEAP-1 POLYNUCLEOTIDES

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a STEAP-1 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a STEAP-1-related protein and fragments thereof, DNA, RNA,DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to a STEAP-1 gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toa STEAP-1 gene, mRNA, or to a STEAP-1 encoding polynucleotide(collectively, “STEAP-1 polynucleotides”). In all instances whenreferred to in this section, T can also be U in FIG. 2.

Embodiments of a STEAP-1 polynucleotide include: a STEAP-1polynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of STEAP-1 as shown in FIG. 2 wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. For example,embodiments of STEAP-1 nucleotides comprise, without limitation:

-   -   (I) a polynucleotide comprising, consisting essentially of, or        consisting of a sequence as shown in FIG. 2, wherein T can also        be U;    -   (II) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2A, from nucleotide        residue number 66 through nucleotide residue number 1085,        including the stop codon, wherein T can also be U;    -   (III) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2B, from nucleotide        residue number 96 through nucleotide residue number 872,        including the stop codon, wherein T can also be U;    -   (IV) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2C, from nucleotide        residue number 96 through nucleotide residue number 944,        including the a stop codon, wherein T can also be U;    -   (V) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2D, from nucleotide        residue number 96 through nucleotide residue number 872,        including the stop codon, wherein T can also be U;    -   (VI) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2E, from nucleotide        residue number 96 through nucleotide residue number 872,        including the stop codon, wherein T can also be U;    -   (VII) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2F, from nucleotide        residue number 96 through nucleotide residue number 872,        including the stop codon, wherein T can also be U;    -   (VIII) a polynucleotide comprising, consisting essentially of,        or consisting of the sequence as shown in FIG. 2G, from        nucleotide residue number 96 through nucleotide residue number        872, including the stop codon, wherein T can also be U;    -   (IX) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2H, from nucleotide        residue number 96 through nucleotide residue number 872,        including the stop codon, wherein T can also be U;    -   (X) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2I, from nucleotide        residue number 96 through nucleotide residue number 872,        including the stop codon, wherein T can also be U;    -   (XI) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2J, from nucleotide        residue number 96 through nucleotide residue number 872,        including the stop codon, wherein T can also be U;    -   (XII) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2K, from nucleotide        residue number 96 through nucleotide residue number 872,        including the stop codon, wherein T can also be U;    -   (XIII) a polynucleotide comprising, consisting essentially of,        or consisting of the sequence as shown in FIG. 2L, from        nucleotide residue number 96 through nucleotide residue number        872, including the stop codon, wherein T can also be U;    -   (XIV) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2M, from nucleotide        residue number 96 through nucleotide residue number 872;        including the stop codon, wherein T can also be U;    -   (XV) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2N, from nucleotide        residue number 96 through nucleotide residue number 872,        including the stop codon, wherein T can also be U;    -   (XVI) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2O, from nucleotide        residue number 96 through nucleotide residue number 872,        including the stop codon, wherein T can also be U;    -   (XVII) a polynucleotide comprising, consisting essentially of,        or consisting of the sequence as shown in FIG. 2P, from        nucleotide residue number 96 through nucleotide residue number        872, including the stop codon, wherein T can also be U;    -   (XVIII) a polynucleotide comprising, consisting essentially of,        or consisting of the sequence as shown in FIG. 2Q, from        nucleotide residue number 96 through nucleotide residue number        872, including the stop codon, wherein T can also be U;    -   (XIX) a polynucleotide that encodes a STEAP-1-related protein        that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%        homologous to an entire amino acid sequence shown in FIG. 2A-Q;    -   (XX) a polynucleotide that encodes a STEAP-1-related protein        that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%        identical to an entire amino acid sequence shown in FIG. 2A-Q;    -   (XXI) a polynucleotide that encodes at least one peptide set        forth in Tables V-XVIII and XXII-LI as set forth in U.S. patent        application Ser. No. 10/236,878 filed 6 Sep. 2002, the specific        contents of which are fully incorporated by reference herein;    -   (XXII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3A in any whole number increment up        to 339 that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (XXIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3A in any whole number increment up        to 339 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XXIV) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3A in any whole number increment up        to 339 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Percent Accessible Residues profile of        FIG. 7;    -   (XXV) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3A in any whole number increment up        to 399 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XXVI) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3A in any whole number increment up        to 339 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Beta-turn profile of FIG. 9;    -   (XXVII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIGS. 3B and 3D in any whole number        increment up to 258 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having        a value greater than 0.5 in the Hydrophilicity profile of FIG.        5;    -   (XXVIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIGS. 3B and 3D in any whole number        increment up to 258 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having        a value less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XXIX) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIGS. 3B and 3D in any whole number        increment up to 258 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having        a value greater than 0.5 in the Percent Accessible Residues        profile of FIG. 7;    -   (XXX) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIGS. 3B and 3D in any whole number        increment up to 258 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having        a value greater than 0.5 in the Average Flexibility profile of        FIG. 8;    -   (XXXI) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIGS. 3B and 3D in any whole number        increment up to 258 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having        a value greater than 0.5 in the Beta-turn profile of FIG. 9;    -   (XXXII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3C in any whole number increment up        to 282 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (XXXIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3C in any whole number increment up        to 282 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XXXIV) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3C in any whole number increment up        to 282 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Percent Accessible Residues profile of        FIG. 7;    -   (XXXV) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3C in any whole number increment up        to 282 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XXXVI) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3C in any whole number increment up        to 282 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Beta-turn profile of FIG. 9;    -   (XXXVII) a polynucleotide that is fully complementary to a        polynucleotide of any one of (I)-(XXXVI);    -   (XXXVIII) a polynucleotide that is fully complementary to a        polynucleotide of any one of (I)-(XXXVII);    -   (XXXIX) a peptide that is encoded by any of (I) to (XXXVIII);        and;    -   (XL) a composition comprising a polynucleotide of any of        (I)-(XXXVIII) or peptide of (XXXIX) together with a        pharmaceutical excipient and/or in a human unit dose form;    -   (XLI) a method of using a polynucleotide of any (I)-(XXXVIII) or        peptide of (XXXIX) or a composition of (XL) in a method to        modulate a cell expressing STEAP-1;    -   (XLII) a method of using a polynucleotide of any (I)-(XXXVIII)        or peptide of (XXXIX) or a composition of (XL) in a method to        diagnose, prophylax, prognose, or treat an individual who bears        a cell expressing STEAP-1;    -   (XLIII) a method of using a polynucleotide of any (I)-(XXXVIII)        or peptide of (XXXIX) or a composition of (XL) in a method to        diagnose, prophylax, prognose, or treat an individual who bears        a cell expressing STEAP-1, said cell from a cancer of a tissue        listed in Table I;    -   (XLIV) a method of using a polynucleotide of any (I)-(XXXVIII)        or peptide of (XXXIX) or a composition of (XL) in a method to        diagnose, prophylax, prognose, or treat a cancer;    -   (XLV) a method of using a polynucleotide of any (I)-(XXXVIII) or        peptide of (XXXIX) or a composition of (XL) in a method to        diagnose, prophylax, prognose, or treat a cancer of a tissue        listed in Table I; and;    -   (XLVI) a method of using a polynucleotide of any (I)-(XXXVIII)        or peptide of (XXXIX) or a composition of (XL) in a method to        identify or characterize a modulator of a cell expressing        STEAP-1.

As used herein, a range is understood to disclose specifically all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include STEAP-1polynucleotides that encode specific portions of STEAP-1 mRNA sequences(and those which are complementary to such sequences) such as those thatencode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 330, 335,339 or more contiguous amino acids of STEAP-1 variant 1; the maximallengths relevant for other variants are: variant 2, 258 amino acids;variant 3, 282 amino acids, and variant 4, 258 amino acids.

For example, representative embodiments of the invention disclosedherein include: polynucleotides and their encoded peptides themselvesencoding about amino acid 1 to about amino acid 10 of the STEAP-1protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 10 to about amino acid 20 of the STEAP-1 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 20 to about amino acid30 of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 30 to about amino acid 40 of the STEAP-1protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 40 to about amino acid 50 of the STEAP-1 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 50 to about amino acid60 of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 60 to about amino acid 70 of the STEAP-1protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 70 to about amino acid 80 of the STEAP-1 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 80 to about amino acid90 of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 90 to about amino acid 100 of the STEAP-1protein shown in FIG. 2 or FIG. 3, in increments of about 10 aminoacids, ending at the carboxyl terminal amino acid set forth in FIG. 2 orFIG. 3. Accordingly, polynucleotides encoding portions of the amino acidsequence (of about 10 amino acids), of amino acids, 100 through thecarboxyl terminal amino acid of the STEAP-1 protein are embodiments ofthe invention. Wherein it is understood that each particular amino acidposition discloses that position plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of a STEAP-1 proteinare also within the scope of the invention. For example, polynucleotidesencoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 30, or 40 or 50 etc.) of the STEAP-1 protein “or variant”shown in FIG. 2 or FIG. 3 can be generated by a variety of techniqueswell known in the art. These polynucleotide fragments can include anyportion of the STEAP-1 sequence as shown in FIG. 2.

Additional illustrative embodiments of the invention disclosed hereininclude STEAP-1 polynucleotide fragments encoding one or more of thebiological motifs contained within a STEAP-1 protein “or variant”sequence, including one or more of the motif-bearing subsequences of aSTEAP-1 protein “or variant” set forth in Tables V-XVIII and XXII-LI. Inanother embodiment, typical polynucleotide fragments of the inventionencode one or more of the regions of STEAP-1 protein or variant thatexhibit homology to a known molecule. In another embodiment of theinvention, typical polynucleotide fragments can encode one or more ofthe STEAP-1 protein or variant N-glycosylation sites, cAMP andcGMP-dependent protein kinase phosphorylation sites, casein kinase IIphosphorylation sites or N-myristoylation site and amidation sites.

Note that to determine the starting position of any peptide set forth inTables V-XVIII and Tables XXII to LI (collectively HLA Peptide Tables)respective to its parental protein, e.g., variant 1, variant 2, etc.,reference is made to three factors: the particular variant, the lengthof the peptide in an HLA Peptide Table, and the Search Peptides listedin Table LII. Generally, a unique Search Peptide is used to obtain HLApeptides for a particular variant. The position of each Search Peptiderelative to its respective parent molecule is listed in Table LII.Accordingly, if a Search Peptide begins at position “X”, one must addthe value “X minus 1” to each position in Tables V-XVIII and TablesXXII-LI to obtain the actual position of the HLA peptides in theirparental molecule. For example if a particular Search Peptide begins atposition 150 of its parental molecule, one must add 150-1, i.e., 149 toeach HLA peptide amino acid position to calculate the position of thatamino acid in the parent molecule.

II.A.) Uses of STEAP-1 Polynucleotides

II.A.1. Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human STEAP-1 gene maps to the chromosomallocation set forth in the Example entitled “Chromosomal Mapping ofSTEAP-1.” For example, because the STEAP-1 gene maps to this chromosome,polynucleotides that encode different regions of the STEAP-1 proteinsare used to characterize cytogenetic abnormalities of this chromosomallocale, such as abnormalities that are identified as being associatedwith various cancers. In certain genes, a variety of chromosomalabnormalities including rearrangements have been identified as frequentcytogenetic abnormalities in a number of different cancers (see e.g.Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al.,Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23):9158-9162 (1988)). Thus, polynucleotides encoding specific regions ofthe STEAP-1 proteins provide new tools that can be used to delineate,with greater precision than previously possible, cytogeneticabnormalities in the chromosomal region that encodes STEAP-1 that maycontribute to the malignant phenotype. In this context, thesepolynucleotides satisfy a need in the art for expanding the sensitivityof chromosomal screening in order to identify more subtle and lesscommon chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet.Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as STEAP-1 was shown to be highly expressed in prostate andother cancers, STEAP-1 polynucleotides are used in methods assessing thestatus of STEAP-1 gene products in normal versus cancerous tissues.Typically, polynucleotides that encode specific regions of the STEAP-1proteins are used to assess the presence of perturbations (such asdeletions, insertions, point mutations, or alterations resulting in aloss of an antigen etc.) in specific regions of the STEAP-1 gene, suchas regions containing one or more motifs. Exemplary assays include bothRT-PCR assays as well as single-strand conformation polymorphism (SSCP)analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378(1999), both of which utilize polynucleotides encoding specific regionsof a protein to examine these regions within the protein.

II.A.2. Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of STEAP-1. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives that specifically bind DNA or RNA in a basepair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the STEAP-1 polynucleotides andpolynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,STEAP-1. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The STEAP-1 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(0-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention can beprepared by treatment of the corresponding 0-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990).Additional STEAP-1 antisense oligonucleotides of the present inventioninclude morpholino antisense oligonucleotides known in the art (see,e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development6: 169-175).

The STEAP-1 antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3′ codons of aSTEAP-1 genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to STEAP-1 mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, STEAP-1 antisense oligonucleotides of the presentinvention are 15 to 30-mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to STEAP-1 mRNA. Optionally, STEAP-1antisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof STEAP-1. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of STEAP-1 expression, see, e.g., L.A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

II.A.3. Primers and Primer Pairs

Further specific embodiments of these nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a STEAP-1 polynucleotide in a sample and as ameans for detecting a cell expressing a STEAP-1 protein.

Examples of such probes include polypeptides comprising all or part ofthe human STEAP-1 cDNA sequence shown in FIG. 2. Examples of primerpairs capable of specifically amplifying STEAP-1 mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta STEAP-1 mRNA.

The STEAP-1 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the STEAP-1 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of STEAP-1 polypeptides; as tools formodulating or inhibiting the expression of the STEAP-1 gene(s) and/ortranslation of the STEAP-1 transcript(s); and as therapeutic agents.

The present invention includes the use of any probe as described hereinto identify and isolate a STEAP-1 or STEAP-1 related nucleic acidsequence from a naturally occurring source, such as humans or othermammals, as well as the isolated nucleic acid sequence per se, whichwould comprise all or most of the sequences found in the probe used.

II.A.4. Isolation of STEAP-1-Encoding Nucleic Acid Molecules

The STEAP-1 cDNA sequences described herein enable the isolation ofother polynucleotides encoding STEAP-1 gene product(s), as well as theisolation of polynucleotides encoding STEAP-1 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms of aSTEAP-1 gene product as well as polynucleotides that encode analogs ofSTEAP-1-related proteins. Various molecular cloning methods that can beemployed to isolate full length cDNAs encoding a STEAP-1 gene are wellknown (see, for example, Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989;Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley andSons, 1995). For example, lambda phage cloning methodologies can beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing STEAP-1gene cDNAs can be identified by probing with a labeled STEAP-1 cDNA or afragment thereof. For example, in one embodiment, a STEAP-1 cDNA (e.g.,FIG. 2) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full-length cDNAs corresponding to a STEAP-1gene. A STEAP-1 gene itself can be isolated by screening genomic DNAlibraries, bacterial artificial chromosome libraries (BACs), yeastartificial chromosome libraries (YACs), and the like, with STEAP-1 DNAprobes or primers.

II.A.5. Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga STEAP-1 polynucleotide, a fragment, analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs,BACs, as well as various viral and non-viral vectors well known in theart, and cells transformed or transfected with such recombinant DNA orRNA molecules. Methods for generating such molecules are well known(see, for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a STEAP-1 polynucleotide, fragment,analog or homologue thereof within a suitable prokaryotic or eukaryotichost cell. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell or aninsect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPr1, other transfectableor transducible prostate cancer cell lines, primary cells (PrEC), aswell as a number of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of STEAP-1or a fragment, analog or homolog thereof can be used to generate STEAP-1proteins or fragments thereof using any number of host-vector systemsroutinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression ofSTEAP-1 proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRokneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, STEAP-1 can be expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1, NIH373 and TsuPr1. The host-vector systems of the invention are useful forthe production of a STEAP-1 protein or fragment thereof. Suchhost-vector systems can be employed to study the functional propertiesof STEAP-1 and STEAP-1 mutations or analogs.

Recombinant human STEAP-1 protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding a STEAP-1-related nucleotide. For example, 293T cells can betransfected with an expression plasmid encoding STEAP-1 or fragment,analog or homolog thereof, a STEAP-1-related protein is expressed in the293T cells, and the recombinant STEAP-1 protein is isolated usingstandard purification methods (e.g., affinity purification usinganti-STEAP-1 antibodies). In another embodiment, a STEAP-1 codingsequence is subcloned into the retroviral vector pSRaαSVtkneo and usedto infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 andrat-1 in order to establish STEAP-1 expressing cell lines. Various otherexpression systems well known in the art can also be employed.Expression constructs encoding a leader peptide joined in frame to aSTEAP-1 coding sequence can be used for the generation of a secretedform of recombinant STEAP-1 protein.

As discussed herein, redundancy in the genetic code permits variation inSTEAP-1 gene sequences. In particular, it is known in the art thatspecific host species often have specific codon preferences, and thusone can adapt the disclosed sequence as preferred for a desired host.For example, preferred analog codon sequences typically have rare codons(i.e., codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the INTERNET such as at URLdna.affrc.go.jp/˜nakamura/codon.html.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080(1989) Skilled artisans understand that the general rule that eukaryoticribosomes initiate translation exclusively at the 5′ proximal AUG codonis abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7):2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

III.) STEAP-1-RELATED PROTEINS

Another aspect of the present invention provides STEAP-1-relatedproteins. Specific embodiments of STEAP-1 proteins comprise apolypeptide having all or part of the amino acid sequence of humanSTEAP-1 as shown in FIG. 2 or FIG. 3, preferably FIG. 2A. Alternatively,embodiments of STEAP-1 proteins comprise variant, homolog or analogpolypeptides that have alterations in the amino acid sequence of STEAP-1shown in FIG. 2 or FIG. 3.

Embodiments of a STEAP-1 polypeptide include: a STEAP-1 polypeptidehaving a sequence shown in FIG. 2, a peptide sequence of a STEAP-1 asshown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of apolypeptide having the sequence as shown in FIG. 2; or, at least 10contiguous peptides of a polypeptide having the sequence as shown inFIG. 2 where T is U. For example, embodiments of STEAP-1 peptidescomprise, without limitation:

-   -   (I) a protein comprising, consisting essentially of, or        consisting of an amino acid sequence as shown in FIG. 2A-Q or        FIG. 3A-D;    -   (II) a STEAP-1-related protein that is at least 90, 91, 92, 93,        94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino        acid sequence shown in FIG. 2A-Q or 3A-D;    -   (III) a STEAP-1-related protein that is at least 90, 91, 92, 93,        94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid        sequence shown in FIG. 2A-Q or 3A-D;    -   (IV) a protein that comprises at least one peptide set forth in        Tables V to LI as set forth in U.S. patent application Ser. No.        10/236,878 filed 6 Sep. 2002 the specific contents of which are        fully incorporated by reference herein, optionally with a        proviso that it is not an entire protein of FIG. 2;    -   (V) a protein that comprises at least one peptide set forth in        Tables V-XVIII, collectively, which peptide is also set forth in        Tables XXII to LI, collectively, optionally with a proviso that        it is not an entire protein of FIG. 2;    -   (VI) a protein that comprises at least two peptides selected        from the peptides set forth in Tables V-LI, optionally with a        proviso that it is not an entire protein of FIG. 2;    -   (VII) a protein that comprises at least two peptides selected        from the peptides set forth in Tables V to LI collectively, with        a proviso that the protein is not a contiguous sequence from an        amino acid sequence of FIG. 2;    -   (VIII) a protein that comprises at least one peptide selected        from the peptides set forth in Tables V-XVIII; and at least one        peptide selected from the peptides set forth in Tables XXII to        LI, with a proviso that the protein is not a contiguous sequence        from an amino acid sequence of FIG. 2;    -   (IX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3A in any whole number increment up to 339 respectively that        includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35 amino acid position(s) having a value greater        than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (X) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A        in any whole number increment up to 339 respectively that        includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3A in any whole number increment up to 339 respectively that        includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Percent Accessible Residues        profile of FIG. 7;    -   (XII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3A in any whole number increment up to 339 respectively that        includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Average Flexibility profile of        FIG. 8;    -   (XIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3A        in any whole number increment up to 339 respectively that        includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Beta-turn profile of FIG. 9;    -   (XIV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3B or 3D, in any whole number increment up to 258 respectively        that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (XV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3B or 3D, in any whole number increment up to 258 respectively        that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having        a value less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XVI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3B or 3D, in any whole number increment up to 258 respectively        that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having        a value greater than 0.5 in the Percent Accessible Residues        profile of FIG. 7;    -   (XVII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3B or 3D, in any whole number increment up to 258 respectively        that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having        a value greater than 0.5 in the Average Flexibility profile of        FIG. 8;    -   (XVIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3B        or 3D in any whole number increment up to 258 respectively that        includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Beta-turn profile of FIG. 9;    -   (XIX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3C, in any whole number increment up to 282 respectively that        includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35 amino acid position(s) having a value greater        than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (XX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3C, in any whole number increment up to 282 respectively that        includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XXI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3C, in any whole number increment up to 282 respectively that        includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Percent Accessible Residues        profile of FIG. 7;    -   (XXII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3C, in any whole number increment up to 282 respectively that        includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Average Flexibility profile of        FIG. 8;    -   (XXIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3C        in any whole number increment up to 282 respectively that        includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Beta-turn profile of FIG. 9;    -   (XXIV) a peptide that occurs at least twice in Tables V-XVIII        and XXII to LI, collectively;    -   (XXV) a peptide that occurs at least three times in Tables        VI-XVIII and XXII to LI, collectively;    -   (XXVI) a peptide that occurs at least four times in Tables        V-XXVIII and XXII to LI, collectively;    -   (XXVII) a peptide that occurs at least five times in Tables        V-XVIII and XXII to LI, collectively;    -   (XXVIII) a peptide that occurs at least once in Tables V-XVIII,        and at least once in tables XXII to LI;    -   (XXIX) a peptide that occurs at least once in Tables V-XVIII,        and at least twice in tables XXII to LI;    -   (XXX) a peptide that occurs at least twice in Tables V-XVIII,        and at least once in tables XXII to LI;    -   (XXXI) a peptide that occurs at least twice in Tables V-XVIII,        and at least twice in tables XXII to LI;    -   (XXXII) a peptide which comprises one two, three, four, or five        of the following characteristics, or an oligonucleotide encoding        such peptide:        -   i) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or greater than            0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in            the Hydrophilicity profile of FIG. 5;        -   ii) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or less than            0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in            the Hydropathicity profile of FIG. 6;        -   iii) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or greater than            0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in            the Percent Accessible Residues profile of FIG. 7;        -   iv) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or greater than            0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in            the Average Flexibility profile of FIG. 8; or,        -   v) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or greater than            0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in            the Beta-turn profile of FIG. 9;    -   (XXXIII) a composition comprising a peptide of (I)-(XXXII) or an        antibody or binding region thereof together with a        pharmaceutical excipient and/or in a human unit dose form.    -   (XXXIV) a method of using a peptide of (I)-(XXXII), or an        antibody or binding region thereof or a composition of (XXXIII)        in a method to modulate a cell expressing STEAP-1;    -   (XXXV) a method of using a peptide of (I)-(XXXII) or an antibody        or binding region thereof or a composition of (XXXIII) in a        method to diagnose, prophylax, prognose, or treat an individual        who bears a cell expressing STEAP-1;    -   (XXXVI) a method of using a peptide of (I)-(XXXII) or an        antibody or binding region thereof or a composition (XXXIII) in        a method to diagnose, prophylax, prognose, or treat an        individual who bears a cell expressing STEAP-1, said cell from a        cancer of a tissue listed in Table I;    -   (XXXVII) a method of using a peptide of (I)-(XXXII) or an        antibody or binding region thereof or a composition of (XXXIII)        in a method to diagnose, prophylax, prognose, or treat a cancer;    -   (XXXVIII) a method of using a peptide of (I)-(XXXII) or an        antibody or binding region thereof or a composition of (XXXIII)        in a method to diagnose, prophylax, prognose, or treat a cancer        of a tissue listed in Table I; and;    -   (XXXIX) a method of using a peptide of (I)-(XXXII) or an        antibody or binding region thereof or a composition (XXXIII) in        a method to identify or characterize a modulator of a cell        expressing STEAP-1;

As used herein, a range is understood to specifically disclose all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include STEAP-1polynucleotides that encode specific portions of STEAP-1 mRNA sequences(and those which are complementary to such sequences) such as those thatencode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 330, 335,339 or more contiguous amino acids of STEAP-1 variant 1; the maximallengths relevant for other variants are: variant 2, 258 amino acids;variant 3, 282 amino acids, variant 4, 258 amino acids.

In general, naturally occurring allelic variants of human STEAP-1 sharea high degree of structural identity and homology (e.g., 90% or morehomology). Typically, allelic variants of a STEAP-1 protein containconservative amino acid substitutions within the STEAP-1 sequencesdescribed herein or contain a substitution of an amino acid from acorresponding position in a homologue of STEAP-1. One class of STEAP-1allelic variants are proteins that share a high degree of homology withat least a small region of a particular STEAP-1 amino acid sequence, butfurther contain a radical departure from the sequence, such as anon-conservative substitution, truncation, insertion or frame shift. Incomparisons of protein sequences, the terms, similarity, identity, andhomology each have a distinct meaning as appreciated in the field ofgenetics. Moreover, orthology and paralogy can be important conceptsdescribing the relationship of members of a given protein family in oneorganism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative aminoacid substitutions can frequently be made in a protein without alteringeither the conformation or the function of the protein. Proteins of theinvention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15conservative substitutions.

Embodiments of the invention disclosed herein include a wide variety ofart-accepted variants or analogs of STEAP-1 proteins such aspolypeptides having amino acid insertions, deletions and substitutions.STEAP-1 variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selectionmutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)) or other known techniques can be performed on the cloned DNA toproduce the STEAP-1 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yieldadequate amounts of variant, an isosteric amino acid can be used.

As defined herein, STEAP-1 variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope that is “crossreactive” with a STEAP-1 protein having an amino acid sequence of FIG.3. As used in this sentence, “cross reactive” means that an antibody orT cell that specifically binds to a STEAP-1 variant also specificallybinds to a STEAP-1 protein having an amino acid sequence set forth inFIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG.3, when it no longer contains any epitope capable of being recognized byan antibody or T cell that specifically binds to the starting STEAP-1protein. Those skilled in the art understand that antibodies thatrecognize proteins bind to epitopes of varying size, and a grouping ofthe order of about four or five amino acids, contiguous or not, isregarded as a typical number of amino acids in a minimal epitope. See,e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955; Hebbes et al.,Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985)135(4):2598-608.

Other classes of STEAP-1-related protein variants share 70%, 75%, 80%,85% or 90% or more similarity with an amino acid sequence of FIG. 3, ora fragment thereof. Another specific class of STEAP-1 protein variantsor analogs comprises one or more of the STEAP-1 biological motifsdescribed herein or presently known in the art. Thus, encompassed by thepresent invention are analogs of STEAP-1 fragments (nucleic or aminoacid) that have altered functional (e.g. immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the full amino acid sequence of aSTEAP-1 protein shown in FIG. 2 or FIG. 3. For example, representativeembodiments of the invention comprise peptides/proteins having any 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of aSTEAP-1 protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed hereininclude polypeptides consisting of about amino acid 1 to about aminoacid 10 of a STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 10 to about amino acid 20 of a STEAP-1protein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 20 to about amino acid 30 of a STEAP-1 protein shown in FIG.2 or FIG. 3, polypeptides consisting of about amino acid 30 to aboutamino acid 40 of a STEAP-1 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofa STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 50 to about amino acid 60 of a STEAP-1 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 toabout amino acid 70 of a STEAP-1 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 70 to about amino acid 80 ofa STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 80 to about amino acid 90 of a STEAP-1 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 toabout amino acid 100 of a STEAP-1 protein shown in FIG. 2 or FIG. 3,etc. throughout the entirety of a STEAP-1 amino acid sequence. Moreover,polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.)to about amino acid 20, (or 130, or 140 or 150 etc.) of a STEAP-1protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. Itis to be appreciated that the starting and stopping positions in thisparagraph refer to the specified position as well as that position plusor minus 5 residues.

STEAP-1-related proteins are generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the art.Alternatively, recombinant methods can be used to generate nucleic acidmolecules that encode a STEAP-1-related protein. In one embodiment,nucleic acid molecules provide a means to generate defined fragments ofa STEAP-1 protein (or variants, homologs or analogs thereof).

III.A.) MOTIF-BEARING PROTEIN EMBODIMENTS

Additional illustrative embodiments of the invention disclosed hereininclude STEAP-1 polypeptides comprising the amino acid residues of oneor more of the biological motifs contained within a STEAP-1 polypeptidesequence set forth in FIG. 2 or FIG. 3. Various motifs are known in theart, and a protein can be evaluated for the presence of such motifs by anumber of publicly available Internet sites (see, e.g., URL addresses:pfam.wustl.edu/;searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html;psort.ims.u-tokyo.acjp/; cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html;expasy.ch/tools/scnpsitl.html; Epimatrix™ and Epimer™, Brown University,brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS,bimas.dcrt.nih.gov/.).

Motif bearing subsequences of all STEAP-1 variant proteins are set forthand identified in Tables V-XVIII and XXII-LI.

Table IV(h) sets forth several frequently occurring motifs based on pfamsearches (see URL address pfam.wustl.edu/). The columns of Table IV(h)list (1) motif name abbreviation, (2) percent identity found amongst thedifferent member of the motif family, (3) motif name or description and(4) most common function; location information is included if the motifis relevant for location.

Polypeptides comprising one or more of the STEAP-1 motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the STEAP-1 motifsdiscussed above are associated with growth dysregulation and becauseSTEAP-1 is overexpressed in certain cancers (See, e.g., Table I). Caseinkinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C,for example, are enzymes known to be associated with the development ofthe malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2):165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995);Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterzielet al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2):305-309 (1998)). Moreover, both glycosylation and myristoylation areprotein modifications also associated with cancer and cancer progression(see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999);Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation isanother protein modification also associated with cancer and cancerprogression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr.(13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified in accordance with art-acceptedmethods, such as the peptides set forth in Tables V-XVIII and XXII-LI.CTL epitopes can be determined using specific algorithms to identifypeptides within a STEAP-1 protein that are capable of optimally bindingto specified HLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, BrownUniversity, URL brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html;and BIMAS, URL bimas.dcrt.nih.gov/.) Moreover, processes for identifyingpeptides that have sufficient binding affinity for HLA molecules andwhich are correlated with being immunogenic epitopes, are well known inthe art, and are carried out without undue experimentation. In addition,processes for identifying peptides that are immunogenic epitopes, arewell known in the art, and are carried out without undue experimentationeither in vitro or in vivo.

Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI and HLA Class II motifs/supermotifs of Table IV). The epitope isanaloged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition. For example, on the basis of residues defined in Table IV, onecan substitute out a deleterious residue in favor of any other residue,such as a preferred residue; substitute a less-preferred residue with apreferred residue; or substitute an originally-occurring preferredresidue with another preferred residue. Substitutions can occur atprimary anchor positions or at other positions in a peptide; see, e.g.,Table IV.

A variety of references reflect the art regarding the identification andgeneration of epitopes in a protein of interest as well as analogsthereof. See, for example, WO 97/33602 to Chesnut et al.; Sette,Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondoet al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol.1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt etal., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7(1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3):266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J.Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9):751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the invention include polypeptides comprisingcombinations of the different motifs set forth in Table(s) IV(a), IV(b),IV(c), IV(d), and IV(h), and/or, one or more of the predicted CTLepitopes of Tables V-XVIII and XXII-LI, and/or, one or more of thepredicted HR epitopes of Tables XLVIII-LI, and/or, one or more of the Tcell binding motifs known in the art. Preferred embodiments contain noinsertions, deletions or substitutions either within the motifs orwithin the intervening sequences of the polypeptides. In addition,embodiments which include a number of either N-terminal and/orC-terminal amino acid residues on either side of these motifs may bedesirable (to, for example, include a greater portion of the polypeptidearchitecture in which the motif is located). Typically, the number ofN-terminal and/or C-terminal amino acid residues on either side of amotif is between about 1 to about 100 amino acid residues, preferably 5to about 50 amino acid residues.

STEAP-1-related proteins are embodied in many forms, preferably inisolated form. A purified STEAP-1 protein molecule will be substantiallyfree of other proteins or molecules that impair the binding of STEAP-1to antibody, T cell or other ligand. The nature and degree of isolationand purification will depend on the intended use. Embodiments of aSTEAP-1-related proteins include purified STEAP-1-related proteins andfunctional, soluble STEAP-1-related proteins. In one embodiment, afunctional, soluble STEAP-1 protein or fragment thereof retains theability to be bound by antibody, T cell or other ligand.

The invention also provides STEAP-1 proteins comprising biologicallyactive fragments of a STEAP-1 amino acid sequence shown in FIG. 2 orFIG. 3. Such proteins exhibit properties of the starting STEAP-1protein, such as the ability to elicit the generation of antibodies thatspecifically bind an epitope associated with the starting STEAP-1protein; to be bound by such antibodies; to elicit the activation of HTLor CTL; and/or, to be recognized by HTL or CTL that also specificallybind to the starting protein.

STEAP-1-related polypeptides that contain particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or based on immunogenicity. Fragments thatcontain such structures are particularly useful in generatingsubunit-specific anti-STEAP-1 antibodies or T cells or in identifyingcellular factors that bind to STEAP-1. For example, hydrophilicityprofiles can be generated, and immunogenic peptide fragments identified,using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can begenerated, and immunogenic peptide fragments identified, using themethod of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol.157:105-132. Percent (%) Accessible Residues profiles can be generated,and immunogenic peptide fragments identified, using the method of JaninJ., 1979, Nature 277:491-492. Average Flexibility profiles can begenerated, and immunogenic peptide fragments identified, using themethod of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. ProteinRes. 32:242-255. Beta-turn profiles can be generated, and immunogenicpeptide fragments identified, using the method of Deleage, G., Roux B.,1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identifypeptides within a STEAP-1 protein that are capable of optimally bindingto specified HLA alleles (e.g., by using the SYFPEITHI site at WorldWide Web URL syfpeithi.bmi-heidelberg.com/; the listings in TableIV(A)-(E); Epimatrix™ and Epimer™, Brown University, URL(brown.edu/ResearchiTB-HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URLbimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from STEAP-1that are presented in the context of human MHC Class I molecules, e.g.,HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., TablesV-XVIII, XXII-LI). Specifically, the complete amino acid sequence of theSTEAP-1 protein and relevant portions of other variants, i.e., for HLAClass I predictions 9 flanking residues on either side of a pointmutation or exon junction, and for HLA Class II predictions 14 flankingresidues on either side of a point mutation or exon junctioncorresponding to that variant, were entered into the HLA Peptide MotifSearch algorithm found in the Bioinformatics and Molecular AnalysisSection (BIMAS) web site listed above; in addition to the siteSYFPEITHI, at URL syfpeithi.bmi-heidelberg.com/.

The HLA peptide motif search algorithm was developed by Dr. Ken Parkerbased on binding of specific peptide sequences in the groove of HLAClass I molecules, in particular HLA-A2 (see, e.g., Falk et al., Nature351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker etal., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol.152:163-75 (1994)). This algorithm allows location and ranking of 8-mer,9-mer, and 10-mer peptides from a complete protein sequence forpredicted binding to HLA-A2 as well as numerous other HLA Class Imolecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers.For example, for Class I HLA-A2, the epitopes preferably contain aleucine (L) or methionine (M) at position 2 and a valine (V) or leucine(L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7(1992)). Selected results of STEAP-1 predicted binding peptides areshown in Tables V-XVIII and XXII-LI herein. In Tables V-XVIII andXXII-XLVIII, selected candidates, 9-mers and 10-mers, for each familymember are shown along with their location, the amino acid sequence ofeach specific peptide, and an estimated binding score. In TablesXLVIII-LI, selected candidates, 15-mers, for each family member areshown along with their location, the amino acid sequence of eachspecific peptide, and an estimated binding score. The binding scorecorresponds to the estimated half time of dissociation of complexescontaining the peptide at 37° C. at pH 6.5. Peptides with the highestbinding score are predicted to be the most tightly bound to HLA Class Ion the cell surface for the greatest period of time and thus representthe best immunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa etal., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides canbe evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

It is to be appreciated that every epitope predicted by the BIMAS site,Epimer™ and Epimatrix™ sites, or specified by the HLA class I or classII motifs available in the art or which become part of the art such asset forth in Table IV (or determined using World Wide Web site URLsyfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be“applied” to a STEAP-1 protein in accordance with the invention. As usedin this context “applied” means that a STEAP-1 protein is evaluated,e.g., visually or by computer-based patterns finding methods, asappreciated by those of skill in the relevant art. Every subsequence ofa STEAP-1 protein of 8, 9, 10, or 11 amino acid residues that bears anHLA Class I motif, or a subsequence of 9 or more amino acid residuesthat bear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of STEAP-1-Related Proteins

In an embodiment described in the examples that follow, STEAP-1 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding STEAP-1 with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted STEAP-1 protein intransfected cells. The secreted HIS-tagged STEAP-1 in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

III.C.) Modifications of STEAP-1-Related Proteins

Modifications of STEAP-1-related proteins such as covalent modificationsare included within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of a STEAP-1polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues of aSTEAP-1 protein. Another type of covalent modification of a STEAP-1polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of a protein of the invention.Another type of covalent modification of STEAP-1 comprises linking aSTEAP-1 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The STEAP-1-related proteins of the present invention can also bemodified to form a chimeric molecule comprising STEAP-1 fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantiy. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments of aSTEAP-1 sequence (amino or nucleic acid) such that a molecule is createdthat is not, through its length, directly homologous to the amino ornucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimericmolecule can comprise multiples of the same subsequence of STEAP-1. Achimeric molecule can comprise a fusion of a STEAP-1-related proteinwith a polyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind, with cytokines or with growthfactors. The epitope tag is generally placed at the amino- orcarboxyl-terminus of a STEAP-1 protein. In an alternative embodiment,the chimeric molecule can comprise a fusion of a STEAP-1-related proteinwith an immunoglobulin or a particular region of an immunoglobulin. Fora bivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of a STEAP-1polypeptide in place of at least one variable region within an Igmolecule. In a preferred embodiment, the immunoglobulin fusion includesthe hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of anIgGI molecule. For the production of immunoglobulin fusions see, e.g.,U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

III.D.) Uses of STEAP-1-Related Proteins

The proteins of the invention have a number of different specific uses.As STEAP-1 is highly expressed in prostate and other cancers,STEAP-1-related proteins are used in methods that assess the status ofSTEAP-1 gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of a STEAP-1 protein are used to assess the presence ofperturbations (such as deletions, insertions, point mutations etc.) inthose regions (such as regions containing one or more motifs). Exemplaryassays utilize antibodies or T cells targeting STEAP-1-related proteinscomprising the amino acid residues of one or more of the biologicalmotifs contained within a STEAP-1 polypeptide sequence in order toevaluate the characteristics of this region in normal versus canceroustissues or to elicit an immune response to the epitope. Alternatively,STEAP-1-related proteins that contain the amino acid residues of one ormore of the biological motifs in a STEAP-1 protein are used to screenfor factors that interact with that region of STEAP-1.

STEAP-1 protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of aSTEAP-1 protein), for identifying agents or cellular factors that bindto STEAP-1 or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

Proteins encoded by the STEAP-1 genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to a STEAP-1 gene product.Antibodies raised against a STEAP-1 protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of STEAP-1protein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. STEAP-1-related nucleic acids or proteins are also used ingenerating HTL or CTL responses.

Various immunological assays useful for the detection of STEAP-1proteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting STEAP-1-expressingcells (e.g., in radioscintigraphic imaging methods). STEAP-1 proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

IV.) STEAP-1 Antibodies

Another aspect of the invention provides antibodies that bind toSTEAP-1-related proteins. Preferred antibodies specifically bind to aSTEAP-1-related protein and do not bind (or bind weakly) to peptides orproteins that are not STEAP-1-related proteins under physiologicalconditions. In this context, examples of physiological conditionsinclude: 1) phosphate buffered saline; 2) Tris-buffered salinecontaining 25 mM Tris and 150 mM NaCl; or normal saline (0.9% NaCl); 4)animal serum such as human serum; or, 5) a combination of any of 1)through 4); these reactions preferably taking place at pH 7.5,alternatively in a range of pH 7.0 to 8.0, or alternatively in a rangeof pH 6.5 to 8.5; also, these reactions taking place at a temperaturebetween 4° C. to 37° C. For example, antibodies that bind STEAP-1 canbind STEAP-1-related proteins such as the homologs or analogs thereof.

STEAP-1 antibodies of the invention are particularly useful in cancer(see, e.g., Table I) diagnostic and prognostic assays, and imagingmethodologies. Similarly, such antibodies are useful in the treatment,diagnosis, and/or prognosis of prostate and other cancers, to the extentSTEAP-1 is also expressed or overexpressed in these other cancers.Moreover, intracellularly expressed antibodies (e.g., single chainantibodies) are therapeutically useful in treating cancers in which theexpression of STEAP-1 is involved, such as advanced or metastaticprostate cancers or other advanced or metastatic cancers.

The invention also provides various immunological assays useful for thedetection and quantification of STEAP-1 and mutant STEAP-1-relatedproteins. Such assays can comprise one or more STEAP-1 antibodiescapable of recognizing and binding a STEAP-1-related protein, asappropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cellimmunogenicity assays (inhibitory or stimulatory) as well as majorhistocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostatecancer and other cancers expressing STEAP-1 are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled STEAP-1 antibodies. Such assays are clinicallyuseful in the detection, monitoring, and prognosis of STEAP-1 expressingcancers such as prostate cancer.

STEAP-1 antibodies are also used in methods for purifying aSTEAP-1-related protein and for isolating STEAP-1 homologues and relatedmolecules. For example, a method of purifying a STEAP-1-related proteincomprises incubating a STEAP-1 antibody, which has been coupled to asolid matrix, with a lysate or other solution containing aSTEAP-1-related protein under conditions that permit the STEAP-1antibody to bind to the STEAP-1-related protein; washing the solidmatrix to eliminate impurities; and eluting the STEAP-1-related proteinfrom the coupled antibody. Other uses of STEAP-1 antibodies inaccordance with the invention include generating anti-idiotypicantibodies that mimic a STEAP-1 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using a STEAP-1-related protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). In addition, fusion proteins of STEAP-1 canalso be used, such as a STEAP-1 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the aminoacid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogento generate appropriate antibodies. In another embodiment, aSTEAP-1-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified STEAP-1-related protein or STEAP-1 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of a STEAP-1 protein as shown in FIG. 2 or FIG.3 can be analyzed to select specific regions of the STEAP-1 protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of a STEAP-1 amino acid sequence are used to identifyhydrophilic regions in the STEAP-1 structure. Regions of a STEAP-1protein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe art, such as Chou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can begenerated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc.Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can begenerated using the method of Kyte, J. and Doolittle, R. F., 1982, J.Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can begenerated using the method of Janin J., 1979, Nature 277:491-492.Average Flexibility profiles can be generated using the method ofBhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res.32:242-255. Beta-turn profiles can be generated using the method ofDeleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, eachregion identified by any of these programs or methods is within thescope of the present invention. Preferred methods for the generation ofSTEAP-1 antibodies are further illustrated by way of the examplesprovided herein. Methods for preparing a protein or polypeptide for useas an immunogen are well known in the art. Also well known in the artare methods for preparing immunogenic conjugates of a protein with acarrier, such as BSA, KLH or other carrier protein. In somecircumstances, direct conjugation using, for example, carbodiimidereagents are used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., are effective.Administration of a STEAP-1 immunogen is often conducted by injectionover a suitable time period and with use of a suitable adjuvant, as isunderstood in the art. During the immunization schedule, titers ofantibodies can be taken to determine adequacy of antibody formation.

STEAP-1 monoclonal antibodies can be produced by various means wellknown in the art. For example, immortalized cell lines that secrete adesired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a STEAP-1-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, byrecombinant means. Regions that bind specifically to the desired regionsof a STEAP-1 protein can also be produced in the context of chimeric orcomplementarity-determining region (CDR) grafted antibodies of multiplespecies origin. Humanized or human STEAP-1 antibodies can also beproduced, and are preferred for use in therapeutic contexts. Methods forhumanizing murine and other non-human antibodies, by substituting one ormore of the non-human antibody CDRs for corresponding human antibodysequences, are well known (see for example, Jones et al., 1986, Nature321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen etal., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc.Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151:2296.

Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535-539). Fully human STEAP-1 monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system: human antibodiesfrom phage display libraries. In: Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, HumanAntibodies from combinatorial libraries. Id., pp 65-82). Fully humanSTEAP-1 monoclonal antibodies can also be produced using transgenic miceengineered to contain human immunoglobulin gene loci as described in PCTPatent Application WO98/24893, Kucherlapati and Jakobovits et al.,published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued 19 Dec. 2000; U.S.Pat. No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No. 6,114,598issued 5 Sep. 2000). This method avoids the in vitro manipulationrequired with phage display technology and efficiently produces highaffinity authentic human antibodies.

Reactivity of STEAP-1 antibodies with a STEAP-1-related protein can beestablished by a number of well-known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,STEAP-1-related proteins, STEAP-1-expressing cells or extracts thereof.A STEAP-1 antibody or fragment thereof can be labeled with a detectablemarker or conjugated to a second molecule. Suitable detectable markersinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme. Further, bi-specific antibodies specific for two or moreSTEAP-1 epitopes are generated using methods generally known in the art.Homodimeric antibodies can also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

In one embodiment, the invention provides for monoclonal antibodiesidentified as mouse hybridoma X92.1.30.1.1(1) and mouse hybridomaX120.545.1.1 deposited with the American Type Culture Collection,located at 10801 University Blvd. Manassas, Va. 20110-2209 on 6 Feb.2004 and assigned ATCC Accession numbers PTA-5802 and PTA-5803respectively.

V.) STEAP-1 CELLULAR IMMUNE RESPONSES

The mechanism by which T cells recognize antigens has been delineated.Efficacious peptide epitope vaccine compositions of the invention inducea therapeutic or prophylactic immune responses in very broad segments ofthe world-wide population. For an understanding of the value andefficacy of compositions of the invention that induce cellular immuneresponses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071,1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. andBodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev.Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are set forth in Table IV (see also, e.g., Southwood, etal., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web atURL (134.2.96.221/scripts.hlaserver.dll/home.htm); Sette, A. and Sidney,J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin.Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol.4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994;Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol.155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996;Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J.Immunogenetics 1999 November; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexeshave revealed pockets within the peptide binding cleft/groove of HLAmolecules which accommodate, in an allele-specific mode, residues borneby peptide ligands; these residues in turn determine the HLA bindingcapacity of the peptides in which they are present. (See, e.g., Madden,D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203,1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H.et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci.USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927,1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol.Biol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs, or class I or class II supermotifs allows identificationof regions within a protein that are correlated with binding toparticular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates forepitope-based vaccines have been identified; such candidates can befurther evaluated by HLA-peptide binding assays to determine bindingaffinity and/or the time period of association of the epitope and itscorresponding HLA molecule. Additional confirmatory work can beperformed to select, amongst these vaccine candidates, epitopes withpreferred characteristics in terms of population coverage, and/orimmunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity,including:

1) Evaluation of primary T cell cultures from normal individuals (see,e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. etal., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J.Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1,1998). This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected using, e.g., a lymphokine- or ⁵¹Cr-release assayinvolving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. etal., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol.8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). Forexample, in such methods peptides in incomplete Freund's adjuvant areadministered subcutaneously to HLA transgenic mice. Several weeksfollowing immunization, splenocytes are removed and cultured in vitro inthe presence of test peptide for approximately one week.Peptide-specific T cells are detected using, e.g., a⁵¹Cr-release assayinvolving peptide sensitized target cells and target cells expressingendogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals whohave been either effectively vaccinated and/or from chronically illpatients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995;Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin.Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648,1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly,recall responses are detected by culturing PBL from subjects that havebeen exposed to the antigen due to disease and thus have generated animmune response “naturally”, or from patients who were vaccinatedagainst the antigen. PBL from subjects are cultured in vitro for 1-2weeks in the presence of test peptide plus antigen presenting cells(APC) to allow activation of “memory” T cells, as compared to “naive” Tcells. At the end of the culture period, T cell activity is detectedusing assays including ⁵¹Cr release involving peptide-sensitizedtargets, T cell proliferation, or lymphokine release.

VI.) STEAP-1 TRANSGENIC ANIMALS

Nucleic acids that encode a STEAP-1-related protein can also be used togenerate either transgenic animals or “knock out” animals that, in turn,are useful in the development and screening of therapeutically usefulreagents. In accordance with established techniques, cDNA encodingSTEAP-1 can be used to clone genomic DNA that encodes STEAP-1. Thecloned genomic sequences can then be used to generate transgenic animalscontaining cells that express DNA that encode STEAP-1. Methods forgenerating transgenic animals, particularly animals such as mice orrats, have become conventional in the art and are described, forexample, in U.S. Pat. No. 4,736,866 issued 12 Apr. 1988, and U.S. Pat.No. 4,870,009 issued 26 Sep. 1989. Typically, particular cells would betargeted for STEAP-1 transgene incorporation with tissue-specificenhancers.

Transgenic animals that include a copy of a transgene encoding STEAP-1can be used to examine the effect of increased expression of DNA thatencodes STEAP-1. Such animals can be used as tester animals for reagentsthought to confer protection from, for example, pathological conditionsassociated with its overexpression. In accordance with this aspect ofthe invention, an animal is treated with a reagent and a reducedincidence of a pathological condition, compared to untreated animalsthat bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

Alternatively, non-human homologues of STEAP-1 can be used to constructa STEAP-1 “knock out” animal that has a defective or altered geneencoding STEAP-1 as a result of homologous recombination between theendogenous gene encoding STEAP-1 and altered genomic DNA encodingSTEAP-1 introduced into an embryonic cell of the animal. For example,cDNA that encodes STEAP-1 can be used to clone genomic DNA encodingSTEAP-1 in accordance with established techniques. A portion of thegenomic DNA encoding STEAP-1 can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic sterncell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of a STEAP-1 polypeptide.

VII.) METHODS FOR THE DETECTION OF STEAP-1

Another aspect of the present invention relates to methods for detectingSTEAP-1 polynucleotides and STEAP-1-related proteins, as well as methodsfor identifying a cell that expresses STEAP-1. The expression profile ofSTEAP-1 makes it a diagnostic marker for metastasized disease.Accordingly, the status of STEAP-1 gene products provides informationuseful for predicting a variety of factors including susceptibility toadvanced stage disease, rate of progression, and/or tumoraggressiveness. As discussed in detail herein, the status of STEAP-1gene products in patient samples can be analyzed by a variety protocolsthat are well known in the art including immunohistochemical analysis,the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

More particularly, the invention provides assays for the detection ofSTEAP-1 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable STEAP-1 polynucleotides include, for example, a STEAP-1gene or fragment thereof, STEAP-1 mRNA, alternative splice variantSTEAP-1 mRNAs, and recombinant DNA or RNA molecules that contain aSTEAP-1 polynucleotide. A number of methods for amplifying and/ordetecting the presence of STEAP-1 polynucleotides are well known in theart and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a STEAP-1 mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using aSTEAP-1 polynucleotides as sense and antisense primers to amplifySTEAP-1 cDNAs therein; and detecting the presence of the amplifiedSTEAP-1 cDNA. Optionally, the sequence of the amplified STEAP-1 cDNA canbe determined.

In another embodiment, a method of detecting a STEAP-1 gene. in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using STEAP-1 polynucleotides assense and antisense primers; and detecting the presence of the amplifiedSTEAP-1 gene. Any number of appropriate sense and antisense probecombinations can be designed from a STEAP-1 nucleotide sequence (see,e.g., FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of aSTEAP-1 protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a STEAP-1-related protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, Westernblot analysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, a method of detecting the presence of a STEAP-1-related proteinin a biological sample comprises first contacting the sample with aSTEAP-1 antibody, a STEAP-1-reactive fragment thereof, or a recombinantprotein containing an antigen-binding region of a STEAP-1 antibody; andthen detecting the binding of STEAP-1-related protein in the sample.

Methods for identifying a cell that expresses STEAP-1 are also withinthe scope of the invention. In one embodiment, an assay for identifyinga cell that expresses a STEAP-1 gene comprises detecting the presence ofSTEAP-1 mRNA in the cell. Methods for the detection of particular mRNAsin cells are well known and include, for example, hybridization assaysusing complementary DNA probes (such as in situ hybridization usinglabeled STEAP-1 riboprobes, Northern blot and related techniques) andvarious nucleic acid amplification assays (such as RT-PCR usingcomplementary primers specific for STEAP-1, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like). Alternatively, an assay for identifying a cell that expressesa STEAP-1 gene comprises detecting the presence of STEAP-1-relatedprotein in the cell or secreted by the cell. Various methods for thedetection of proteins are well known in the art and are employed for thedetection of STEAP-1-related proteins and cells that expressSTEAP-1-related proteins.

STEAP-1 expression analysis is also useful as a tool for identifying andevaluating agents that modulate STEAP-1 gene expression. For example,STEAP-1 expression is significantly upregulated in prostate cancer, andis expressed in cancers of the tissues listed in Table I. Identificationof a molecule or biological agent that inhibits STEAP-1 expression orover-expression in cancer cells is of therapeutic value. For example,such an agent can be identified by using a screen that quantifiesSTEAP-1 expression by RT-PCR, nucleic acid hybridization or antibodybinding.

VIII.) METHODS FOR MONITORING THE STATUS OF STEAP-1-RELATED GENES ANDTHEIR PRODUCTS

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,Cancer Surv. 23: 19-32 (1995)). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrantSTEAP-1 expression in cancers) allows for early detection of suchaberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of STEAP-1 in abiological sample of interest can be compared, for example, to thestatus of STEAP-1 in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not affectedby a pathology). An alteration in the status of STEAP-1 in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not affected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Greyer et al., J. Comp. Neurol.1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compareSTEAP-1 status in a sample.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of STEAP-1 expressing cells) as well as the level, andbiological activity of expressed gene products (such as STEAP-1 mRNA,polynucleotides and polypeptides). Typically, an alteration in thestatus of STEAP-1 comprises a change in the location of STEAP-1 and/orSTEAP-1 expressing cells and/or an increase in STEAP-1 mRNA and/orprotein expression.

STEAP-1 status in a sample can be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of a STEAP-1 geneand gene products are found, for example in Ausubel et al. eds., 1995,Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4(Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus,the status of STEAP-1 in a biological sample is evaluated by variousmethods utilized by skilled artisans including, but not limited togenomic Southern analysis (to examine, for example perturbations in aSTEAP-1 gene), Northern analysis and/or PCR analysis of STEAP-1 mRNA (toexamine, for example alterations in the polynucleotide sequences orexpression levels of STEAP-1 mRNAs), and, Western and/orimmunohistochemical analysis (to examine, for example alterations inpolypeptide sequences, alterations in polypeptide localization within asample, alterations in expression levels of STEAP-1 proteins and/orassociations of STEAP-1 proteins with polypeptide binding partners).Detectable STEAP-1 polynucleotides include, for example, a STEAP-1 geneor fragment thereof, STEAP-1 mRNA, alternative splice variants, STEAP-1mRNAs, and recombinant DNA or RNA molecules containing a STEAP-1polynucleotide.

The expression profile of STEAP-1 makes it a diagnostic marker for localand/or metastasized disease, and provides information on the growth oroncogenic potential of a biological sample. In particular, the status ofSTEAP-1 provides information useful for predicting susceptibility toparticular disease stages, progression, and/or tumor aggressiveness. Theinvention provides methods and assays for determining STEAP-1 status anddiagnosing cancers that express STEAP-1, such as cancers of the tissueslisted in Table I. For example, because STEAP-1 mRNA is so highlyexpressed in prostate and other cancers relative to normal prostatetissue, assays that evaluate the levels of STEAP-1 mRNA transcripts orproteins in a biological sample can be used to diagnose a diseaseassociated with STEAP-1 dysregulation, and can provide prognosticinformation useful in defining appropriate therapeutic options.

The expression status of STEAP-1 provides information including thepresence, stage and location of dysplastic, precancerous and cancerouscells, predicting susceptibility to various stages of disease, and/orfor gauging tumor aggressiveness. Moreover, the expression profile makesit useful as an imaging reagent for metastasized disease. Consequently,an aspect of the invention is directed to the various molecularprognostic and diagnostic methods for examining the status of STEAP-1 inbiological samples such as those from individuals suffering from, orsuspected of suffering from a pathology characterized by dysregulatedcellular growth, such as cancer.

As described above, the status of STEAP-1 in a biological sample can beexamined by a number of well-known procedures in the art. For example,the status of STEAP-1 in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of STEAP-1 expressing cells (e.g. those that expressSTEAP-1 mRNAs or proteins). This examination can provide evidence ofdysregulated cellular growth, for example, when STEAP-1-expressing cellsare found in a biological sample that does not normally contain suchcells (such as a lymph node), because such alterations in the status ofSTEAP-1 in a biological sample are often associated with dysregulatedcellular growth. Specifically, one indicator of dysregulated cellulargrowth is the metastases of cancer cells from an organ of origin (suchas the prostate) to a different area of the body (such as a lymph node).In this context, evidence of dysregulated cellular growth is importantfor example because occult lymph node metastases can be detected in asubstantial proportion of patients with prostate cancer, and suchmetastases are associated with known predictors of disease progression(see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000); Su et al.,Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995August 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring STEAP-1gene products by determining the status of STEAP-1 gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of STEAP-1gene products in a corresponding normal sample. The presence of aberrantSTEAP-1 gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in STEAP-1 mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of STEAP-1 mRNA can, for example, beevaluated in tissues including but not limited to those listed in TableI. The presence of significant STEAP-1 expression in any of thesetissues is useful to indicate the emergence, presence and/or severity ofa cancer, since the corresponding normal tissues do not express STEAP-1mRNA or express it at lower levels.

In a related embodiment, STEAP-1 status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodcomprises determining the level of STEAP-1 protein expressed by cells ina test tissue sample and comparing the level so determined to the levelof STEAP-1 expressed in a corresponding normal sample. In oneembodiment, the presence of STEAP-1 protein is evaluated, for example,using immunohistochemical methods. STEAP-1 antibodies or bindingpartners capable of detecting STEAP-1 protein expression are used in avariety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of STEAP-1nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions and thelike. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, amutation in the sequence of STEAP-1 may be indicative of the presence orpromotion of a tumor. Such assays therefore have diagnostic andpredictive value where a mutation in STEAP-1 indicates a potential lossof function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of STEAP-1 geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S.Pat. No. 5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of a STEAP-1 genein a biological sample. Aberrant demethylation and/or hypermethylationof CpG islands in gene 5′ regulatory regions frequently occurs inimmortalized and transformed cells, and can result in altered expressionof various genes. For example, promoter hypermethylation of the pi-classglutathione S-transferase (a protein expressed in normal prostate butnot expressed in >90% of prostate carcinomas) appears to permanentlysilence transcription of this gene and is the most frequently detectedgenomic alteration in prostate carcinomas (De Matzo et al., Am. J.Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration ispresent in at least 70% of cases of high-grade prostatic intraepithelialneoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarters Prev.,1998, 7:531-536). In another example, expression of the LAGE-I tumorspecific gene (which is not expressed in normal prostate but isexpressed in 25-50% of prostate cancers) is induced by deoxy-azacytidinein lymphoblastoid cells, suggesting that tumoral expression is due todemethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). Avariety of assays for examining methylation status of a gene are wellknown in the art. For example, one can utilize, in Southernhybridization approaches, methylation-sensitive restriction enzymes thatcannot cleave sequences that contain methylated CpG sites to assess themethylation status of CpG islands. In addition, MSP (methylationspecific PCR) can rapidly profile the methylation status of all the CpGsites present in a CpG island of a given gene. This procedure involvesinitial modification of DNA by sodium bisulfite (which will convert allunmethylated cytosines to uracil) followed by amplification usingprimers specific for methylated versus unmethylated DNA. Protocolsinvolving methylation interference can also be found for example inCurrent Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel etal. eds., 1995.

Gene amplification is an additional method for assessing the status ofSTEAP-1. Gene amplification is measured in a sample directly, forexample, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for thepresence of cancer cells using for example, Northern, dot blot or RT-PCRanalysis to detect STEAP-1 expression. The presence of RT-PCRamplifiable STEAP-1 mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art. RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.In the prostate cancer field, these include RT-PCR assays for thedetection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol.Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibilitythat an individual has for developing cancer. In one embodiment, amethod for predicting susceptibility to cancer comprises detectingSTEAP-1 mRNA or STEAP-1 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of STEAP-1 mRNAexpression correlates to the degree of susceptibility. In a specificembodiment, the presence of STEAP-1 in prostate or other tissue isexamined, with the presence of STEAP-1 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). Similarly, one can evaluate theintegrity STEAP-1 nucleotide and amino acid sequences in a biologicalsample, in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like. Thepresence of one or more perturbations in STEAP-1 gene products in thesample is an indication of cancer susceptibility (or the emergence orexistence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness.In one embodiment, a method for gauging aggressiveness of a tumorcomprises determining the level of STEAP-1 mRNA or STEAP-1 proteinexpressed by tumor cells, comparing the level so determined to the levelof STEAP-1 mRNA or STEAP-1 protein expressed in a corresponding normaltissue taken from the same individual or a normal tissue referencesample, wherein the degree of STEAP-1 mRNA or STEAP-1 protein expressionin the tumor sample relative to the normal sample indicates the degreeof aggressiveness. In a specific embodiment, aggressiveness of a tumoris evaluated by determining the extent to which STEAP-1 is expressed inthe tumor cells, with higher expression levels indicating moreaggressive tumors. Another embodiment is the evaluation of the integrityof STEAP-1 nucleotide and amino acid sequences in a biological sample,in order to identify perturbations in the structure of these moleculessuch as insertions, deletions, substitutions and the like. The presenceof one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observingthe progression of a malignancy in an individual over time. In oneembodiment, methods for observing the progression of a malignancy in anindividual over time comprise determining the level of STEAP-1 mRNA orSTEAP-1 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of STEAP-1 mRNA or STEAP-1 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of STEAP-1 mRNA or STEAP-1protein expression in the tumor sample over time provides information onthe progression of the cancer. In a specific embodiment, the progressionof a cancer is evaluated by determining STEAP-1 expression in the tumorcells over time, where increased expression over time indicates aprogression of the cancer. Also, one can evaluate the integrity STEAP-1nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, where the presence ofone or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention is directed to methods forobserving a coincidence between the expression of STEAP-1 gene andSTEAP-1 gene products (or perturbations in STEAP-1 gene and STEAP-1 geneproducts) and a factor that is associated with malignancy, as a meansfor diagnosing and prognosticating the status of a tissue sample. A widevariety of factors associated with malignancy can be utilized, such asthe expression of genes associated with malignancy (e.g. PSA, PSCA andPSM expression for prostate cancer etc.) as well as gross cytologicalobservations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol.6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al.,1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg.Pathol. 23(8):918-24). Methods for observing a coincidence between theexpression of STEAP-1 gene and STEAP-1 gene products (or perturbationsin STEAP-1 gene and STEAP-1 gene products) and another factor that isassociated with malignancy are useful, for example, because the presenceof a set of specific factors that coincide with disease providesinformation crucial for diagnosing and prognosticating the status of atissue sample.

In one embodiment, methods for observing a coincidence between theexpression of STEAP-1 gene and STEAP-1 gene products (or perturbationsin STEAP-1 gene and STEAP-1 gene products) and another factor associatedwith malignancy entails detecting the overexpression of STEAP-1 mRNA orprotein in a tissue sample, detecting the overexpression of PSA mRNA orprotein in a tissue sample (or PSCA or PSM expression), and observing acoincidence of STEAP-1 mRNA or protein and PSA mRNA or proteinoverexpression (or PSCA or PSM expression). In a specific embodiment,the expression of STEAP-1 and PSA mRNA in prostate tissue is examined,where the coincidence of STEAP-1 and PSA mRNA overexpression in thesample indicates the existence of prostate cancer, prostate cancersusceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of STEAP-1 mRNA orprotein are described herein, and standard nucleic acid and proteindetection and quantification technologies are well known in the art.Standard methods for the detection and quantification of STEAP-1 mRNAinclude in situ hybridization using labeled STEAP-1 riboprobes, Northernblot and related techniques using STEAP-1 polynucleotide probes, RT-PCRanalysis using primers specific for STEAP-1, and other amplificationtype detection methods, such as, for example, branched DNA, SISBA, TMAand the like. In a specific embodiment, semi-quantitative RT-PCR is usedto detect and quantify STEAP-1 mRNA expression. Any number of primerscapable of amplifying STEAP-1 can be used for this purpose, includingbut not limited to the various primer sets specifically describedherein. In a specific embodiment, polyclonal or monoclonal antibodiesspecifically reactive with the wild-type STEAP-1 protein can be used inan immunohistochemical assay of biopsied tissue.

IX.) IDENTIFICATION OF MOLECULES THAT INTERACT WITH STEAP-1

The STEAP-1 protein and nucleic acid sequences disclosed herein allow askilled artisan to identify proteins, small molecules and other agentsthat interact with STEAP-1, as well as pathways activated by STEAP-1 viaany one of a variety of art accepted protocols. For example, one canutilize one of the so-called interaction trap systems (also referred toas the “two-hybrid assay”). In such systems, molecules interact andreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Other systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator, see, e.g.,U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and6,004,746 issued 21 Dec. 1999. Algorithms are also available in the artfor genome-based predictions of protein function (see, e.g., Marcotte,et al., Nature 402: 4 Nov. 1999, 83-86).

Alternatively one can screen peptide libraries to identify moleculesthat interact with STEAP-1 protein sequences. In such methods, peptidesthat bind to STEAP-1 are identified by screening libraries that encode arandom or controlled collection of amino acids. Peptides encoded by thelibraries are expressed as fusion proteins of bacteriophage coatproteins, the bacteriophage particles are then screened against theSTEAP-1 protein(s).

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with STEAP-1 proteinsequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3Mar. 1998 and 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express STEAP-1 are used to identifyprotein-protein interactions mediated by STEAP-1. Such interactions canbe examined using immunoprecipitation techniques (see, e.g., Hamilton B.J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). STEAP-1protein can be immunoprecipitated from STEAP-1-expressing cell linesusing anti-STEAP-1 antibodies. Alternatively, antibodies against His-tagcan be used in a cell line engineered to express fusions of STEAP-1 anda His-tag (vectors mentioned above). The immunoprecipitated complex canbe examined for protein association by procedures such as Westernblotting, ³⁵S-methionine labeling of proteins, protein microsequencing,silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with STEAP-1 can be identifiedthrough related embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with STEAP-1's ability to mediatephosphorylation and de-phosphorylation, interaction with DNA or RNAmolecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate STEAP-1-related ion channel, protein pump, or cellcommunication functions are identified and used to treat patients thathave a cancer that expresses STEAP-1 (see, e.g., Hille, B., IonicChannels of Excitable Membranes 2^(nd) Ed., Sinauer Assoc., Sunderland,Mass., 1992). Moreover, ligands that regulate STEAP-1 function can beidentified based on their ability to bind STEAP-1 and activate areporter construct. Typical methods are discussed for example in U.S.Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forminghybrid ligands in which at least one ligand is a small molecule. In anillustrative embodiment, cells engineered to express a fusion protein ofSTEAP-1 and a DNA-binding protein are used to co-express a fusionprotein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying modulators, which activate or inhibit STEAP-1.

An embodiment of this invention comprises a method of screening for amolecule that interacts with a STEAP-1 amino acid sequence shown in FIG.2 or FIG. 3, comprising the steps of contacting a population ofmolecules with a STEAP-1 amino acid sequence, allowing the population ofmolecules and the STEAP-1 amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the STEAP-1 amino acid sequence, and thenseparating molecules that do not interact with the STEAP-1 amino acidsequence from molecules that do. In a specific embodiment, the methodfurther comprises purifying, characterizing and identifying a moleculethat interacts with the STEAP-1 amino acid sequence. The identifiedmolecule can be used to modulate a function performed by STEAP-1. In apreferred embodiment, the STEAP-1 amino acid sequence is contacted witha library of peptides.

X.) THERAPEUTIC METHODS AND COMPOSITIONS

The identification of STEAP-1 as a protein that is normally expressed ina restricted set of tissues, but which is also expressed in cancers suchas those listed in Table 1, opens a number of therapeutic approaches tothe treatment of such cancers.

Of note, targeted antitumor therapies have been useful even when thetargeted protein is expressed on normal tissues, even vital normal organtissues. A vital organ is one that is necessary to sustain life, such asthe heart or colon. A non-vital organ is one that can be removedwhereupon the individual is still able to survive. Examples of non-vitalorgans are ovary, breast, and prostate.

For example, Herceptin® is an FDA approved pharmaceutical that consistsof an antibody which is immunoreactive with the protein variously knownas HER2, HER2/neu, and erb-b-2. It is marketed by Genentech and has beena commercially successful antitumor agent. Herceptin® sales reachedalmost $400 million in 2002. Herceptin® is a treatment for HER2 positivemetastatic breast cancer. However, the expression of HER2 is not limitedto such tumors. The same protein is expressed in a number of normaltissues. In particular, it is known that HER2/neu is present in normalkidney and heart, thus these tissues are present in all human recipientsof Herceptin. The presence of HER2/neu in normal kidney is alsoconfirmed by Latif, Z., et al., B.J.U. International (2002) 89:5-9. Asshown in this article (which evaluated whether renal cell carcinomashould be a preferred indication for anti-HER2 antibodies such asHerceptin) both protein and mRNA are produced in benign renal tissues.Notably, HER2/neu protein was strongly overexpressed in benign renaltissue. Despite the fact that HER2/neu is expressed in such vitaltissues as heart and kidney, Herceptin is a very useful, FDA approved,and commercially successful drug. The effect of Herceptin on cardiactissue, i.e., “cardiotoxicity,” has merely been a side effect totreatment. When patients were treated with Herceptin alone, significantcardiotoxicity occurred in a very low percentage of patients. Tominimize cariotoxicity there is a more stringent entry requirement forthe treatment with HER2/neu. Factors such as predisposition to heartcondition are evaluated before treatment can occur.

Of particular note, although kidney tissue is indicated to exhibitnormal expression, possibly even higher expression than cardiac tissue,kidney has no appreciable Herceptin side effect whatsoever. Moreover, ofthe diverse array of normal tissues in which HER2 is expressed, there isvery little occurrence of any side effect. Only cardiac tissue hasmanifested any appreciable side effect at all. A tissue such as kidney,where HER2/neu expression is especially notable, has not been the basisfor any side effect.

Furthermore, favorable therapeutic effects have been found for antitumortherapies that target epidermal growth factor receptor (EGFR); Erbitux(ImClone). EGFR is also expressed in numerous normal tissues. There havebeen very limited side effects in normal tissues following use ofanti-EGFR therapeutics. A general side effect that occurs with the EGFRtreatment is a severe skin rash observed in 100% of the patientsundergoing treatment.

Thus, expression of a target protein in normal tissue, even vital normaltissue, does not defeat the utility of a targeting agent for the proteinas a therapeutic for certain tumors in which the protein is alsooverexpressed. For example, expression in vital organs is not in and ofitself detrimental. In addition, organs regarded as dispensible, such asthe prostate and ovary, can be removed without affecting mortality.Finally, some vital organs are not affected by normal organ expressionbecause of an immunoprivilage. Immunoprivilaged organs are organs thatare protected from blood by a blood-organ barrier and thus are notaccessible to immunotherapy. Examples of immunoprivilaged organs are thebrain and testis.

Accordingly, therapeutic approaches that inhibit the activity of aSTEAP-1 protein are useful for patients suffering from a cancer thatexpresses STEAP-1. These therapeutic approaches generally fall intothree classes. The first class modulates STEAP-1 function as it relatesto tumor cell growth leading to inhibition or retardation of tumor cellgrowth or inducing its killing. The second class comprises variousmethods for inhibiting the binding or association of a STEAP-1 proteinwith its binding partner or with other proteins. The third classcomprises a variety of methods for inhibiting the transcription of aSTEAP-1 gene or translation of STEAP-1 mRNA.

X.A.) Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a STEAP-1-relatedprotein or STEAP-1-related nucleic acid. In view of the expression ofSTEAP-1, cancer vaccines prevent and/or treat STEAP-1-expressing cancerswith minimal or no effects on non-target tissues. The use of a tumorantigen in a vaccine that generates cell-mediated humoral immuneresponses as anti-cancer therapy is well known in the art and has beenemployed in prostate cancer using human PSMA and rodent PAP immunogens(Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J.Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a STEAP-1-relatedprotein, or a STEAP-1-encoding nucleic acid molecule and recombinantvectors capable of expressing and presenting the STEAP-1 immunogen(which typically comprises a number of T-cell epitopes or antibody)Skilled artisans understand that a wide variety of vaccine systems fordelivery of immunoreactive epitopes are known in the art (see, e.g.,Heryln et al., Ann Med 1999 Feb. 31(1):66-78; Maruyama et al., CancerImmunol Immunother 2000 June 49(3):123-32) Briefly, such methods ofgenerating an immune response (e.g. cell-mediated and/or humoral) in amammal, comprise the steps of: exposing the mammal's immune system to animmunoreactive epitope (e.g. an epitope present in a STEAP-1 proteinshown in FIG. 3 or analog or homolog thereof) so that the mammalgenerates an immune response that is specific for that epitope (e.g.generates antibodies that specifically recognize that epitope). In apreferred method, a STEAP-1 immunogen contains a biological motif, seee.g., Tables V-XVIII and XXII-LI, or a peptide of a size range fromSTEAP-1 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire STEAP-1 protein, immunogenic regions or epitopes thereof canbe combined and delivered by various means. Such vaccine compositionscan include, for example, lipopeptides (e.g., Vitiello, A. et al., J.Clin. Invest. 95:341, 1995), peptide compositions encapsulated inpoly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge,et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptidecompositions contained in immune stimulating complexes (ISCOMS) (see,e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin ExpImmunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J.P., J. Immunol. Methods 196:17-32, 1996), peptides formulated asmultivalent peptides; peptides for use in ballistic delivery systems,typically crystallized peptides, viral delivery vectors (Perkus, M. E.et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. etal., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda,P. K. et al., Virology 175:535, 1990), particles of viral or syntheticorigin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996;Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. etal., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al.,Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted delivery technologies, also known as receptormediated targeting, such as those of Avant Immunotherapeutics, Inc.(Needham, Mass.) may also be used.

In patients with STEAP-1-associated cancer, the vaccine compositions ofthe invention can also be used in conjunction with other treatments usedfor cancer, e.g., surgery, chemotherapy, drug therapies, radiationtherapies, etc. including use in combination with immune adjuvants suchas IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identifypeptides within STEAP-1 protein that bind corresponding HLA alleles (seee.g., Table IV; Epimer™ and Epimatrix™, Brown University (URLbrown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and, BIMAS,(URL bimas.dcrt.nih.gov/; SYFPEITHI at URLsyfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a STEAP-1immunogen contains one or more amino acid sequences identified usingtechniques well known in the art, such as the sequences shown in TablesV-XVIII and XXII-LI or a peptide of 8, 9, 10 or 11 amino acids specifiedby an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), orTable IV (E)) and/or a peptide of at least 9 amino acids that comprisesan HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)).As is appreciated in the art, the HLA Class I binding groove isessentially closed ended so that peptides of only a particular sizerange can fit into the groove and be bound, generally HLA Class Iepitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLAClass II binding groove is essentially open ended; therefore a peptideof about 9 or more amino acids can be bound by an HLA Class II molecule.Due to the binding groove differences between HLA Class I and II, HLAClass I motifs are length specific, i.e., position two of a Class Imotif is the second amino acid in an amino to carboxyl direction of thepeptide. The amino acid positions in a Class II motif are relative onlyto each other, not the overall peptide, i.e., additional amino acids canbe attached to the amino and/or carboxyl termini of a motif-bearingsequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than25 amino acids.

A wide variety of methods for generating an immune response in a mammalare known in the art (for example as the first step in the generation ofhybridomas). Methods of generating an immune response in a mammalcomprise exposing the mammal's immune system to an immunogenic epitopeon a protein (e.g. a STEAP-1 protein) so that an immune response isgenerated. A typical embodiment consists of a method for generating animmune response to STEAP-1 in a host, by contacting the host with asufficient amount of at least one STEAP-1 B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the STEAP-1 B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a STEAP-1-related protein or aman-made multiepitopic peptide comprising: administering STEAP-1immunogen (e.g. a STEAP-1 protein or a peptide fragment thereof, aSTEAP-1 fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE™ peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92). An alternativemethod comprises generating an immune response in an individual againsta STEAP-1 immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes a STEAP-1 immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a geneticvaccine facilitator such as anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea is also administered. In addition, an antiidiotypic antibody can beadministered that mimics STEAP-1, in order to generate a response to thetarget antigen.

Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediatedmodalities. DNA or RNA that encode protein(s) of the invention can beadministered to a patient. Genetic immunization methods can be employedto generate prophylactic or therapeutic humoral and cellular immuneresponses directed against cancer cells expressing STEAP-1. Constructscomprising DNA encoding a STEAP-1-related protein/immunogen andappropriate regulatory sequences can be injected directly into muscle orskin of an individual, such that the cells of the muscle or skin take-upthe construct and express the encoded STEAP-1 protein/immunogen.Alternatively, a vaccine comprises a STEAP-1-related protein. Expressionof the STEAP-1-related protein immunogen results in the generation ofprophylactic or therapeutic humoral and cellular immunity against cellsthat bear a STEAP-1 protein. Various prophylactic and therapeuticgenetic immunization techniques known in the art can be used (forreview, see information and references published at Internet addressgenweb.com). Nucleic acid-based delivery is described, for instance, inWolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos.5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO98/04720. Examples of DNA-based delivery technologies include “nakedDNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery,cationic lipid complexes, and particle-mediated (“gene gun”) orpressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of theinvention can be expressed via viral or bacterial vectors. Various viralgene delivery systems that can be used in the practice of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol.8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)).Non-viral delivery systems can also be employed by introducing naked DNAencoding a STEAP-1-related protein into the patient (e.g.,intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotidesequences that encode the peptides of the invention. Upon introductioninto a host, the recombinant vaccinia virus expresses the proteinimmunogenic peptide, and thereby elicits a host immune response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover et al.,Nature 351:456-460 (1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

Thus, gene delivery systems are used to deliver a STEAP-1-relatednucleic acid molecule. In one embodiment, the full-length human STEAP-1cDNA is employed. In another embodiment, STEAP-1 nucleic acid moleculesencoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopesare employed.

Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immuneresponse. One approach involves the use of antigen presenting cells(APCs) such as dendritic cells (DC) to present STEAP-1 antigen to apatient's immune system. Dendritic cells express MHC class I and IImolecules, B7 co-stimulator, and IL-12, and are thus highly specializedantigen presenting cells. In prostate cancer, autologous dendritic cellspulsed with peptides of the prostate-specific membrane antigen (PSMA)are being used in a Phase I clinical trial to stimulate prostate cancerpatients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphyet al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used topresent STEAP-1 peptides to T cells in the context of MHC class I or IImolecules. In one embodiment, autologous dendritic cells are pulsed withSTEAP-1 peptides capable of binding to MHC class I and/or class IImolecules. In another embodiment, dendritic cells are pulsed with thecomplete STEAP-1 protein. Yet another embodiment involves engineeringthe overexpression of a STEAP-1 gene in dendritic cells using variousimplementing vectors known in the art, such as adenovirus (Arthur etal., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al.,1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), ortumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177-1182). Cells that express STEAP-1 can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

X.B.) STEAP-1 as a Target for Antibody-Based Therapy

STEAP-1 is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because STEAP-1 is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration ofSTEAP-1-immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of STEAP-1 areuseful to treat STEAP-1-expressing cancers systemically, either asconjugates with a toxin or therapeutic agent, or as naked antibodiescapable of inhibiting cell proliferation or function.

STEAP-1 antibodies can be introduced into a patient such that theantibody binds to STEAP-1 and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of STEAP-1,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis. Examples include Rituxan® for Non-HodgkinsLymphoma, Herceptin® for metastatic breast cancer, and Erbitux® forcolorectal cancer.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of a STEAP-1 sequence shown in FIG. 2 or FIG. 3. Inaddition, skilled artisans understand that it is routine to conjugateantibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:113678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. STEAP-1), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an animal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. an anti-STEAP-1 antibody) that binds to a marker (e.g. STEAP-1)expressed, accessible to binding or localized on the cell surfaces. Atypical embodiment is a method of delivering a cytotoxic and/ortherapeutic agent to a cell expressing STEAP-1, comprising conjugatingthe cytotoxic agent to an antibody that immunospecifically binds to aSTEAP-1 epitope, and, exposing the cell to the antibody-agent conjugate.Another illustrative embodiment is a method of treating an individualsuspected of suffering from metastasized cancer, comprising a step ofadministering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-STEAP-1 antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin or radioisotope, such as the conjugation ofY⁹¹ or I¹³¹ to anti-CD20 antibodies (e.g., Zevalin™, IDECPharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals) respectively,while others involve co-administration of antibodies and othertherapeutic agents, such as Herceptin™ (trastuzuMAb) with paclitaxel(Genentech, Inc.). The antibodies can be conjugated to a therapeuticagent. To treat prostate cancer, for example, STEAP-1 antibodies can beadministered in conjunction with radiation, chemotherapy or hormoneablation. Also, antibodies can be conjugated to a toxin such ascalicheamicin (e.g., Mylotarg™, Wyeth-Ayerst, Madison, N.J., arecombinant humanized IgG₄ kappa antibody conjugated to antitumorantibiotic calicheamicin) or a maytansinoid (e.g., taxane-basedTumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, Mass.,also see e.g., U.S. Pat. No. 5,416,064) or Auristatin E (SeattleGenetics).

Although STEAP-1 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well. Fan et al. (Cancer Res.53:4637-4642, 1993), Prewett et al. (International J. of Onco.9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991)describe the use of various antibodies together with chemotherapeuticagents.

Although STEAP-1 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of STEAP-1expression, preferably using immunohistochemical assessments of tumortissue, quantitative STEAP-1 imaging, or other techniques that reliablyindicate the presence and degree of STEAP-1 expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

Anti-STEAP-1 monoclonal antibodies that treat prostate and other cancersinclude those that initiate a potent immune response against the tumoror those that are directly cytotoxic. In this regard, anti-STEAP-1monoclonal antibodies (MAbs) can elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-STEAP-1 MAbs that exerta direct biological effect on tumor growth are useful to treat cancersthat express STEAP-1. Mechanisms by which directly cytotoxic MAbs actinclude: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-STEAP-1 MAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric MAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target STEAP-1antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-STEAP-1 MAbs as well as combinations, or cocktails, ofdifferent MAbs. Such MAb cocktails can have certain advantages inasmuchas they contain MAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic MAbs with MAbs thatrely on immune effector functionality. Such MAbs in combination canexhibit synergistic therapeutic effects. In addition, anti-STEAP-1 MAbscan be administered concomitantly with other therapeutic modalities,including but not limited to various chemotherapeutic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. The anti-STEAP-1 MAbs are administered in their “naked” orunconjugated form, or can have a therapeutic agent(s) conjugated tothem.

Anti-STEAP-1 antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of the anti-STEAP-1antibody preparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range of about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of10-1000 mg MAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin™ MAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kgIV of the anti-STEAP-1 MAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a90-minute or longer infusion. The periodic maintenance dose isadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. As appreciated by those of skill in the art,various factors can influence the ideal dose regimen in a particularcase. Such factors include, for example, the binding affinity and halflife of the Ab or MAbs used, the degree of STEAP-1 expression in thepatient, the extent of circulating shed STEAP-1 antigen, the desiredsteady-state antibody concentration level, frequency of treatment, andthe influence of chemotherapeutic or other agents used in combinationwith the treatment method of the invention, as well as the health statusof a particular patient.

Optionally, patients should be evaluated for the levels of STEAP-1 in agiven sample (e.g. the levels of circulating STEAP-1 antigen and/orSTEAP-1 expressing cells) in order to assist in the determination of themost effective dosing regimen, etc. Such evaluations are also used formonitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

Anti-idiotypic anti-STEAP-1 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga STEAP-1-related protein. In particular, the generation ofanti-idiotypic antibodies is well known in the art; this methodology canreadily be adapted to generate anti-idiotypic anti-STEAP-1 antibodiesthat mimic an epitope on a STEAP-1-related protein (see, for example,Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin.Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother.43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccinestrategies.

X.C.) STEAP-1 as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more HLA-binding peptides asdescribed herein are further embodiments of the invention. Furthermore,vaccines in accordance with the invention encompass compositions of oneor more of the claimed peptides. A peptide can be present in a vaccineindividually. Alternatively, the peptide can exist as a homopolymercomprising multiple copies of the same peptide, or as a heteropolymer ofvarious peptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTLs thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly _(L)-lysine,poly _(L)-glutamic acid, influenza, hepatitis B virus core protein, andthe like. The vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, preferably phosphatebuffered saline. The vaccines also typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are examples of materials well known in theart. Additionally, as disclosed herein, CTL responses can be primed byconjugating peptides of the invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS). Moreover, anadjuvant such as a syntheticcytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotideshas been found to increase CTL responses 10- to 100-fold. (see, e.g.Davila and Celis, J. Immunol. 165:539-547 (2000))

Upon immunization with a peptide composition in accordance with theinvention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later development of cells thatexpress or overexpress STEAP-1 antigen, or derives at least sometherapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptidecomponents with components that induce or facilitate neutralizingantibody and or helper T cell responses directed to the target antigen.A preferred embodiment of such a composition comprises class I and classII epitopes in accordance with the invention. An alternative embodimentof such a composition comprises a class I and/or class II epitope inaccordance with the invention, along with a cross reactive HTL epitopesuch as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g.,in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells(APC), such as dendritic cells (DC), as a vehicle to present peptides ofthe invention. Vaccine compositions can be created in vitro, followingdendritic cell mobilization and harvesting, whereby loading of dendriticcells occurs in vitro. For example, dendritic cells are transfected,e.g., with a minigene in accordance with the invention, or are pulsedwith peptides. The dendritic cell can then be administered to a patientto elicit immune responses in vivo. Vaccine compositions, either DNA- orpeptide-based, can also be administered in vivo in combination withdendritic cell mobilization whereby loading of dendritic cells occurs invivo.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition for use ina vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles be balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with tumor clearance.For HLA Class I this includes 3-4 epitopes that come from at least onetumor associated antigen (TAA). For HLA Class II a similar rationale isemployed; again 3-4 epitopes are selected from at least one TAA (see,e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAAmay be used in combination with epitopes from one or more additionalTAAs to produce a vaccine that targets tumors with varying expressionpatterns of frequently-expressed TAAs.

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, often 200 nM or less; and for Class II an IC₅₀of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array ofallele-specific motif-bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess the breadth, or redundancyof, population coverage.

4.) When selecting epitopes from cancer-related antigens it is oftenuseful to select analogs because the patient may have developedtolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as ‘nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A nested peptide sequence can comprise B cell,HLA class I and/or HLA class II epitopes. When providing nestedepitopes, a general objective is to provide the greatest number ofepitopes per sequence. Thus, an aspect is to avoid providing a peptidethat is any longer than the amino terminus of the amino terminal epitopeand the carboxyl terminus of the carboxyl terminal epitope in thepeptide. When providing a multi-epitopic sequence, such as a sequencecomprising nested epitopes, it is generally important to screen thesequence in order to insure that it does not have pathological or otherdeleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene,an objective is to generate the smallest peptide that encompasses theepitopes of interest. This principle is similar, if not the same as thatemployed when selecting a peptide comprising nested epitopes. However,with an artificial polyepitopic peptide, the size minimization objectiveis balanced against the need to integrate any spacer sequences betweenepitopes in the polyepitopic protein. Spacer amino acid residues can,for example, be introduced to avoid junctional epitopes (an epitoperecognized by the immune system, not present in the target antigen, andonly created by the man-made juxtaposition of epitopes), or tofacilitate cleavage between epitopes and thereby enhance epitopepresentation. Junctional epitopes are generally to be avoided becausethe recipient may generate an immune response to that non-nativeepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

7.) Where the sequences of multiple variants of the same target proteinare present, potential peptide epitopes can also be selected on thebasis of their conservancy. For example, a criterion for conservancy maydefine that the entire sequence of an HLA class I binding peptide or theentire 9-mer core of a class II binding peptide be conserved in adesignated percentage of the sequences evaluated for a specific proteinantigen.

X.C.1. Minigene Vaccines

A number of different approaches are available which allow simultaneousdelivery of multiple epitopes. Nucleic acids encoding the peptides ofthe invention are a particularly useful embodiment of the invention.Epitopes for inclusion in a minigene are preferably selected accordingto the guidelines set forth in the previous section. A preferred meansof administering nucleic acids encoding the peptides of the inventionuses minigene constructs encoding a peptide comprising one or multipleepitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka etal., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J.Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996;Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine16:426, 1998. For example, a multi-epitope DNA plasmid encodingsupermotif- and/or motif-bearing epitopes derived STEAP-1, the PADRE®universal helper T cell epitope or multiple HTL epitopes from STEAP-1(see e.g., Tables V-XVIII and XXII to LI), and an endoplasmicreticulum-translocating signal sequence can be engineered. A vaccine mayalso comprise epitopes that are derived from other TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed intransgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, antibody epitopes,a ubiquitination signal sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger peptides comprising the epitope(s) are within thescope of the invention.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LelF), costimulatory molecules, orfor HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego,Calif.). Helper (HTL) epitopes can be joined to intracellular targetingsignals and expressed separately from expressed CTL epitopes; thisallows direction of the HTL epitopes to a cell compartment differentthan that of the CTL epitopes. If required, this could facilitate moreefficient entry of HTL epitopes into the HLA class II pathway, therebyimproving HTL induction. In contrast to HTL or CTL induction,specifically decreasing the immune response by co-expression ofimmunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well-knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is currently being used for intramuscular (IM) administration inclinical trials. To maximize the immunotherapeutic effects of minigeneDNA vaccines, an alternative method for formulating purified plasmid DNAmay be desirable. A variety of methods have been described, and newtechniques may become available. Cationic lipids, glycolipids, andfusogenic liposomes can also be used in the formulation (see, e.g., asdescribed by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7):682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al.,Proc. Natl. Acad. Sci. USA 84:7413 (1987). In addition, peptides andcompounds referred to collectively as protective, interactive,non-condensing compounds (PINC) could also be complexed to purifiedplasmid DNA to influence variables such as stability, intramusculardispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS,intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days afterimmunization, splenocytes are harvested and restimulated for one week inthe presence of peptides encoding each epitope being tested. Thereafter,for CTL effector cells, assays are conducted for cytolysis ofpeptide-loaded, ⁵¹Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs. Immunogenicity of HTLepitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

Minigenes can also be delivered using other bacterial or viral deliverysystems well known in the art, e.g., an expression construct encodingepitopes of the invention can be incorporated into a viral vector suchas vaccinia.

X.C.2. Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can bemodified, e.g., analoged, to provide desired attributes, such asimproved serum half life, broadened population coverage or enhancedimmunogenicity.

For instance, the ability of a peptide to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Although a CTL peptide can be directly linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in a majority of a genetically diversepopulation. This can be accomplished by selecting peptides that bind tomany, most, or all of the HLA class II molecules. Examples of such aminoacid bind many HLA Class II molecules include sequences from antigenssuch as tetanus toxoid at positions 830-843 QYIKANSKFIGITE; (SEQ ID NO:64), Plasmodium falciparum circumsporozoite (CS) protein at positions378-398 DIEKKIAKMEKASSVFNVVNS; (SEQ ID NO: 65), and Streptococcus 18kDprotein at positions 116-131 GAVDSILGGVATYGAA; (SEQ ID NO: 66). Otherexamples include peptides bearing a DR 1-4-7 supermotif, or either ofthe DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed, most preferably, to bind most HLA-DR (human HLAclass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: XKXVAAWTLKAAX (SEQ ID NO: 67), where “X” is eithercyclohexylalanine, phenylalanine, or tyrosine, and a is either_(D)-alanine or _(L)-alanine, has been found to bind to most HLA-DRalleles, and to stimulate the response of T helper lymphocytes from mostindividuals, regardless of their HLA type. An alternative of a pan-DRbinding epitope comprises all “L” natural amino acids and can beprovided in the form of nucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biologicalproperties. For example, they can be modified to include _(D)-aminoacids to increase their resistance to proteases and thus extend theirserum half life, or they can be conjugated to other molecules such aslipids, proteins, carbohydrates, and the like to increase theirbiological activity. For example, a T helper peptide can be conjugatedto one or more palmitic acid chains at either the amino or carboxyltermini.

X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primes Blymphocytes or T lymphocytes. Lipids have been identified as agentscapable of priming CTL in vivo. For example, palmitic acid residues canbe attached to the ε- and α-amino groups of a lysine residue and thenlinked, e.g., via one or more linking residues such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidatedpeptide can then be administered either directly in a micelle orparticle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P₃CSS, for example,and the lipopeptide administered to an individual to prime specificallyan immune response to the target antigen. Moreover, because theinduction of neutralizing antibodies can also be primed withP₃CSS-conjugated epitopes, two such compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTLPeptides

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood. Apharmaceutical to facilitate harvesting of DC can be used, such asProgenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4.After pulsing the DC with peptides and prior to reinfusion intopatients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DCs which present thepulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of whichstimulate CTL responses to STEAP-1. Optionally, a helper T cell (HTL)peptide, such as a natural or artificial loosely restricted HLA Class IIpeptide, can be included to facilitate the CTL response. Thus, a vaccinein accordance with the invention is used to treat a cancer whichexpresses or overexpresses STEAP-1.

X.D.) Adoptive Immunotherapy

Antigenic STEAP-1-related peptides are used to elicit a CTL and/or HTLresponse ex vivo, as well. The resulting CTL or HTL cells, can be usedto treat tumors in patients that do not respond to other conventionalforms of therapy, or will not respond to a therapeutic vaccine peptideor nucleic acid in accordance with the invention. Ex vivo CTL or HTLresponses to a particular antigen are induced by incubating in tissueculture the patient's, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells (APC), such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (e.g., atumor cell). Transfected dendritic cells may also be used as antigenpresenting cells.

X.E.) Administration of Vaccines for Therapeutic or ProphylacticPurposes

Pharmaceutical and vaccine compositions of the invention are typicallyused to treat and/or prevent a cancer that expresses or overexpressesSTEAP-1. In therapeutic applications, peptide and/or nucleic acidcompositions are administered to a patient in an amount sufficient toelicit an effective B cell, CTL and/or HTL response to the antigen andto cure or at least partially arrest or slow symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already bearing a tumor that expresses STEAP-1. The peptidesor DNA encoding them can be administered individually or as fusions ofone or more peptide sequences. Patients can be treated with theimmunogenic peptides separately or in conjunction with other treatments,such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the firstdiagnosis of STEAP-1-associated cancer. This is followed by boostingdoses until at least symptoms are substantially abated and for a periodthereafter. The embodiment of the vaccine composition (i.e., including,but not limited to embodiments such as peptide cocktails, polyepitopicpolypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells)delivered to the patient may vary according to the stage of the diseaseor the patients health status. For example, in a patient with a tumorthat expresses STEAP-1, a vaccine comprising STEAP-1-specific CTL may bemore efficacious in killing tumor cells in patient with advanced diseasethan alternative embodiments.

It is generally important to provide an amount of the peptide epitopedelivered by a mode of administration sufficient to stimulateeffectively a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1,000μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.Dosage values for a human typically range from about 500 μg to about50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0μg to about 50,000 μg of peptide pursuant to a boosting regimen overweeks to months may be administered depending upon the patient'sresponse and condition as determined by measuring the specific activityof CTL and HTL obtained from the patient's blood. Administration shouldcontinue until at least clinical symptoms or laboratory tests indicatethat the neoplasia, has been eliminated or reduced and for a periodthereafter. The dosages, routes of administration, and dose schedulesare adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the presentinvention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely asprophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine can be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, nasal, intrathecal, or local (e.g. as acream or topical ointment) administration. Preferably, thepharmaceutical compositions are administered parentally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH-adjusting and buffering agents, tonicity adjusting agents, wettingagents, preservatives, and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sortitanmonolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in apharmaceutical composition that comprises a human unit dose of anacceptable carrier, in one embodiment an aqueous carrier, and isadministered in a volume/quantity that is known by those of skill in theart to be used for administration of such compositions to humans (see,e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro,Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptidedose for initial immunization can be from about 1 to about 50,000 μg,generally 100-5,000 μg, for a 70 kg patient. For example, for nucleicacids an initial immunization may be performed using an expressionvector in the form of naked nucleic acid administered IM (or SC or ID)in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to1000 μg) can also be administered using a gene gun. Following anincubation period of 3-4 weeks, a booster dose is then administered. Thebooster can be recombinant fowlpox virus administered at a dose of 5-10⁷to 5×10⁹ pfu.

For antibodies, a treatment generally involves repeated administrationof the anti-STEAP-1 antibody preparation, via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1 to about 10 mg/kg body weight. In general,doses in the range of 10-500 mg MAb per week are effective and welltolerated. Moreover, an initial loading dose of approximately 4 mg/kgpatient body weight IV, followed by weekly doses of about 2 mg/kg IV ofthe anti-STEAP-1 MAb preparation represents an acceptable dosingregimen. As appreciated by those of skill in the art, various factorscan influence the ideal dose in a particular case. Such factors include,for example, half life of a composition, the binding affinity of an Ab,the immunogenicity of a substance, the degree of STEAP-1 expression inthe patient, the extent of circulating shed STEAP-1 antigen, the desiredsteady-state concentration level, frequency of treatment, and theinfluence of chemotherapeutic or other agents used in combination withthe treatment method of the invention, as well as the health status of aparticular patient. Non-limiting preferred human unit doses are, forexample, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg,800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, thedose is in a range of 2-5 mg/kg body weight, e.g., with follow on weeklydoses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg bodyweight followed, e.g., in two, three or four weeks by weekly doses;0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks byweekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m² of body areaweekly; 1-600 mg m² of body area weekly; 225-400 mg m² of body areaweekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7,8, 9, 19, 11, 12 or more weeks.

In one embodiment, human unit dose forms of polynucleotides comprise asuitable dosage range or effective amount that provides any therapeuticeffect. As appreciated by one of ordinary skill in the art a therapeuticeffect depends on a number of factors, including the sequence of thepolynucleotide, molecular weight of the polynucleotide and route ofadministration. Dosages are generally selected by the physician or otherhealth care professional in accordance with a variety of parametersknown in the art, such as severity of symptoms, history of the patientand the like. Generally, for a polynucleotide of about 20 bases, adosage range may be selected from, for example, an independentlyselected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to anindependently selected upper limit, greater than the lower limit, ofabout 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dosemay be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg,0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg.Generally, parenteral routes of administration may require higher dosesof polynucleotide compared to more direct application to the nucleotideto diseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitabledosage range or effective amount that provides any therapeutic effect.As appreciated by one of ordinary skill in the art, a therapeutic effectdepends on a number of factors. Dosages are generally selected by thephysician or other health care professional in accordance with a varietyof parameters known in the art, such as severity of symptoms, history ofthe patient and the like. A dose may be about 10⁴ cells to about 10⁶cells, about 10⁶ cells to about 10⁸ cells, about 10⁸ to about 10¹¹cells, or about 10⁸ to about 5×10¹⁰ cells. A dose may also about 10⁶cells/m² to about 10¹⁰ cells/m², or about 10⁶ cells/m² to about 10⁸cells/m².

Proteins(s) of the invention, and/or nucleic acids encoding theprotein(s), can also be administered via liposomes, which may also serveto: 1) target the proteins(s) to a particular tissue, such as lymphoidtissue; 2) to target selectively to diseases cells; or, 3) to increasethe half-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepeptide to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to a receptor prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inaccordance with the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidlability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably suppliedin finely divided form along with a surfactant and propellant. Typicalpercentages of peptides are about 0.01%-20% by weight, preferably about1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from about 6 to 22 carbonatoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute about 0.1%-20%by weight of the composition, preferably about 0.25-5%. The balance ofthe composition is ordinarily propellant. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

XI.) DIAGNOSTIC AND PROGNOSTIC EMBODIMENTS OF STEAP-1

As disclosed herein, STEAP-1 polynucleotides, polypeptides, reactivecytotoxic T cells (CTL), reactive helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic,prognostic and therapeutic assays that examine conditions associatedwith dysregulated cell growth such as cancer, in particular the cancerslisted in Table I (see, e.g., both its specific pattern of tissueexpression as well as its overexpression in certain cancers as describedfor example in the Example entitled “Expression analysis of STEAP-1 innormal tissues, and patient specimens”).

STEAP-1 can be analogized to a prostate associated antigen PSA, thearchetypal marker that has been used by medical practitioners for yearsto identify and monitor the presence of prostate cancer (see, e.g.,Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J.Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. CancerInst. 91(19): 1635-1640(1999)). A variety of other diagnostic markersare also used in similar contexts including p53 and K-ras (see, e.g.,Tulchinsky et al., Int J Mol Med 1999 July 4(1):99-102 and Minimoto etal., Cancer Detect Prev 2000; 24(1):1-12). Therefore, this disclosure ofSTEAP-1 polynucleotides and polypeptides (as well as STEAP-1polynucleotide probes and anti-STEAP-1 antibodies used to identify thepresence of these molecules) and their properties allows skilledartisans to utilize these molecules in methods that are analogous tothose used, for example, in a variety of diagnostic assays directed toexamining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the STEAP-1polynucleotides, polypeptides, reactive T cells and antibodies areanalogous to those methods from well-established diagnostic assays,which employ, e.g., PSA polynucleotides, polypeptides, reactive T cellsand antibodies. For example, just as PSA polynucleotides are used asprobes (for example in Northern analysis, see, e.g., Sharief et al.,Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example inPCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190(2000)) to observe the presence and/or the level of PSA mRNAs in methodsof monitoring PSA overexpression or the metastasis of prostate cancers,the STEAP-1 polynucleotides described herein can be utilized in the sameway to detect STEAP-1 overexpression or the metastasis of prostate andother cancers expressing this gene. Alternatively, just as PSApolypeptides are used to generate antibodies specific for PSA which canthen be used to observe the presence and/or the level of PSA proteins inmethods to monitor PSA protein overexpression (see, e.g., Stephan etal., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells(see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), theSTEAP-1 polypeptides described herein can be utilized to generateantibodies for use in detecting STEAP-1 overexpression or the metastasisof prostate cells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the lung or prostate gland etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing STEAP-1polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain STEAP-1-expressing cells (lymph node) is found tocontain STEAP-1-expressing cells such as the STEAP-1 expression seen inLAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively STEAP-1 polynucleotides and/or polypeptides can be used toprovide evidence of cancer, for example, when cells in a biologicalsample that do not normally express STEAP-1 or express STEAP-1 at adifferent level are found to express STEAP-1 or have an increasedexpression of STEAP-1 (see, e.g., the STEAP-1 expression in the cancerslisted in Table I and in patient samples etc. shown in the accompanyingFigures). In such assays, artisans may further wish to generatesupplementary evidence of metastasis by testing the biological samplefor the presence of a second tissue restricted marker (in addition toSTEAP-1) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res.Pract. 192(3): 233-237 (1996)).

The use of immunohistochemistry to identify the presence of a STEAP-1polypeptide within a tissue section can indicate an altered state ofcertain cells within that tissue. It is well understood in the art thatthe ability of an antibody to localize to a polypeptide that isexpressed in cancer cells is a way of diagnosing presence of disease,disease stage, progression and/or tumor aggressiveness. Such an antibodycan also detect an altered distribution of the polypeptide within thecancer cells, as compared to corresponding non-malignant tissue.

The STEAP-1 polypeptide and immunogenic compositions are also useful inview of the phenomena of altered subcellular protein localization indisease states. Alteration of cells from normal to diseased state causeschanges in cellular morphology and is often associated with changes insubcellular protein localization/distribution. For example, cellmembrane proteins that are expressed in a polarized manner in normalcells can be altered in disease, resulting in distribution of theprotein in a non-polar manner over the whole cell surface.

The phenomenon of altered subcellular protein localization in a diseasestate has been demonstrated with MUC1 and Her2 protein expression by useof immunohistochemical means. Normal epithelial cells have a typicalapical distribution of MUC1, in addition to some supranuclearlocalization of the glycoprotein, whereas malignant lesions oftendemonstrate an apolar staining pattern (Diaz et al, The Breast Journal,7; 40-45 (2001); Zhang et al, Clinical Cancer Research, 4; 2669-2676(1998): Cao, et al, The Journal of Histochemistry and Cytochemistry, 45:1547-1557 (1997)). In addition, normal breast epithelium is eithernegative for Her2 protein or exhibits only a basolateral distributionwhereas malignant cells can express the protein over the whole cellsurface (De Potter, et al, International Journal of Cancer, 44; 969-974(1989): McCormick, et al, 117; 935-943 (2002)). Alternatively,distribution of the protein may be altered from a surface onlylocalization to include diffuse cytoplasmic expression in the diseasedstate. Such an example can be seen with MUC1 (Diaz, et al, The BreastJournal, 7: 40-45 (2001)).

Alteration in the localization/distribution of a protein in the cell, asdetected by immunohistochemical methods, can also provide valuableinformation concerning the favorability of certain treatment modalities.This last point is illustrated by a situation where a protein may beintracellular in normal tissue, but cell surface in malignant cells; thecell surface location makes the cells favorably amenable toantibody-based diagnostic and treatment regimens. When such analteration of protein localization occurs for STEAP-1, the STEAP-1protein and immune responses related thereto are very useful.Accordingly, the ability to determine whether alteration of subcellularprotein localization occurred for 24P4C12 make the STEAP-1 protein andimmune responses related thereto very useful. Use of the STEAP-1compositions allows those skilled in the art to make importantdiagnostic and therapeutic decisions.

Immunohistochemical reagents specific to STEAP-1 are also useful todetect metastases of tumors expressing STEAP-1 when the polypeptideappears in tissues where STEAP-1 is not normally produced.

Thus, STEAP-1 polypeptides and antibodies resulting from immuneresponses thereto are useful in a variety of important contexts such asdiagnostic, prognostic, preventative and/or therapeutic purposes knownto those skilled in the art.

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,STEAP-1 polynucleotide fragments and polynucleotide variants are used inan analogous manner. In particular, typical PSA polynucleotides used inmethods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in the Example entitled “Expression analysisof STEAP-1 in normal tissues, and patient specimens,” where a STEAP-1polynucleotide fragment is used as a probe to show the expression ofSTEAP-1 RNAs in cancer cells. In addition, variant polynucleotidesequences are typically used as primers and probes for the correspondingmRNAs in PCR and Northern analyses (see, e.g., Sawai et al., FetalDiagn. Ther. 1996 November-December 11(6):407-13 and Current ProtocolsIn Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al.eds., 1995)). Polynucleotide fragments and variants are useful in thiscontext where they are capable of binding to a target polynucleotidesequence (e.g., a STEAP-1 polynucleotide shown in FIG. 2 or variantthereof) under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can berecognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. STEAP-1 polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubelet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the STEAP-1biological motifs discussed herein or a motif-bearing subsequence whichis readily identified by one of skill in the art based on motifsavailable in the art. Polypeptide fragments, variants or analogs aretypically useful in this context as long as they comprise an epitopecapable of generating an antibody or T cell specific for a targetpolypeptide sequence (e.g. a STEAP-1 polypeptide shown in FIG. 3).

As shown herein, the STEAP-1 polynucleotides and polypeptides (as wellas the STEAP-1 polynucleotide probes and anti-STEAP-1 antibodies or Tcells used to identify the presence of these molecules) exhibit specificproperties that make them useful in diagnosing cancers such as thoselisted in Table I. Diagnostic assays that measure the presence ofSTEAP-1 gene products, in order to evaluate the presence or onset of adisease condition described herein, such as prostate cancer, are used toidentify patients for preventive measures or further monitoring, as hasbeen done so successfully with PSA. Moreover, these materials satisfy aneed in the art for molecules having similar or complementarycharacteristics to PSA in situations where, for example, a definitediagnosis of metastasis of prostatic origin cannot be made on the basisof a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract.192(3): 233-237 (1996)), and consequently, materials such as STEAP-1polynucleotides and polypeptides (as well as the STEAP-1 polynucleotideprobes and anti-STEAP-1 antibodies used to identify the presence ofthese molecules) need to be employed to confirm a metastases ofprostatic origin.

Finally, in addition to their use in diagnostic assays, the STEAP-1polynucleotides disclosed herein have a number of other utilities suchas their use in the identification of oncogenetic associated chromosomalabnormalities in the chromosomal region to which the STEAP-1 gene maps(see the Example entitled “Chromosomal Mapping of STEAP-1” below).Moreover, in addition to their use in diagnostic assays, theSTEAP-1-related proteins and polynucleotides disclosed herein have otherutilities such as their use in the forensic analysis of tissues ofunknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun. 28;80(1-2): 63-9).

Additionally, STEAP-1-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of STEAP-1. For example, the amino acid or nucleicacid sequence of FIG. 2 or FIG. 3, or fragments of either, can be usedto generate an immune response to a STEAP-1 antigen. Antibodies or othermolecules that react with STEAP-1 can be used to modulate the functionof this molecule, and thereby provide a therapeutic benefit.

XII.) INHIBITION OF STEAP-1 PROTEIN FUNCTION

The invention includes various methods and compositions for inhibitingthe binding of STEAP-1 to its binding partner or its association withother protein(s) as well as methods for inhibiting STEAP-1 function.

XII.A.) Inhibition of STEAP-1 with Intracellular Antibodies

In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to STEAP-1 are introduced into STEAP-1expressing cells via gene transfer technologies. Accordingly, theencoded single chain anti-STEAP-1 antibody is expressed intracellularly,binds to STEAP-1 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, arespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatment isfocused. This technology has been successfully applied in the art (forreview, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodieshave been shown to virtually eliminate the expression of otherwiseabundant cell surface receptors (see, e.g., Richardson et al., 1995,Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol.Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well-known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to target precisely the intrabody to thedesired intracellular compartment. For example, intrabodies targeted tothe endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL amino acid motif. Intrabodies intended to exert activity in thenucleus are engineered to include a nuclear localization signal. Lipidmoieties are joined to intrabodies in order to tether the intrabody tothe cytosolic side of the plasma membrane. Intrabodies can also betargeted to exert function in the cytosol. For example, cytosolicintrabodies are used to sequester factors within the cytosol, therebypreventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture STEAP-1 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such STEAP-1 intrabodies in orderto achieve the desired targeting. Such STEAP-1 intrabodies are designedto bind specifically to a particular STEAP-1 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to a STEAP-1protein are used to prevent STEAP-1 from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing STEAP-1 from forming transcription complexeswith other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

XII.B.) Inhibition of STEAP-1 with Recombinant Proteins

In another approach, recombinant molecules bind to STEAP-1 and therebyinhibit STEAP-1 function. For example, these recombinant moleculesprevent or inhibit STEAP-1 from accessing/binding to its bindingpartner(s) or associating with other protein(s). Such recombinantmolecules can, for example, contain the reactive part(s) of a STEAP-1specific antibody molecule. In a particular embodiment, the STEAP-1binding domain of a STEAP-1 binding partner is engineered into a dimericfusion protein, whereby the fusion protein comprises two STEAP-1 ligandbinding domains linked to the Fc portion of a human IgG, such as humanIgGl. Such IgG portion can contain, for example, the C_(H)2 and C_(H)3domains and the hinge region, but not the C_(H)1 domain. Such dimericfusion proteins are administered in soluble form to patients sufferingfrom a cancer associated with the expression of STEAP-1, whereby thedimeric fusion protein specifically binds to STEAP-1 and blocks STEAP-1interaction with a binding partner. Such dimeric fusion proteins arefurther combined into multimeric proteins using known antibody linkingtechnologies.

XII.C.) Inhibition of STEAP-1 Transcription or Translation

The present invention also comprises various methods and compositionsfor inhibiting the transcription of the STEAP-1 gene. Similarly, theinvention also provides methods and compositions for inhibiting thetranslation of STEAP-1 mRNA into protein.

In one approach, a method of inhibiting the transcription of the STEAP-1gene comprises contacting the STEAP-1 gene with a STEAP-1 antisensepolynucleotide. In another approach, a method of inhibiting STEAP-1 mRNAtranslation comprises contacting a STEAP-1 mRNA with an antisensepolynucleotide. In another approach, a STEAP-1 specific ribozyme is usedto cleave a STEAP-1 message, thereby inhibiting translation. Suchantisense and ribozyme based methods can also be directed to theregulatory regions of the STEAP-1 gene, such as STEAP-1 promoter and/orenhancer elements. Similarly, proteins capable of inhibiting a STEAP-1gene transcription factor are used to inhibit STEAP-1 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of STEAP-1 by interferingwith STEAP-1 transcriptional activation are also useful to treat cancersexpressing STEAP-1. Similarly, factors that interfere with STEAP-1processing are useful to treat cancers that express STEAP-1. Cancertreatment methods utilizing such factors are also within the scope ofthe invention.

XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to delivertherapeutic polynucleotide molecules to tumor cells synthesizing STEAP-1(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother STEAP-1 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding STEAP-1 antisensepolynucleotides, ribozymes, factors capable of interfering with STEAP-1transcription, and so forth, can be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays that evaluate therapeutic activity include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of STEAP-1 to a bindingpartner, etc.

In vivo, the effect of a STEAP-1 therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models can be used, wherein human prostate cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine3: 402-408). For example, PCT Patent Application WO98/16628 and U.S.Pat. No. 6,107,540 describe various xenograft models of human prostatecancer capable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy can be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful inevaluating therapeutic compositions. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

XIII.) IDENTIFICATION, CHARACTERIZATION AND USE OF MODULATORS OF STEAP-1

Methods to Identify and Use Modulators

In one embodiment, screening is performed to identify modulators thatinduce or suppress a particular expression profile, suppress or inducespecific pathways, preferably generating the associated phenotypethereby. In another embodiment, having identified differentiallyexpressed genes important in a particular state; screens are performedto identify modulators that alter expression of individual genes, eitherincrease or decrease. In another embodiment, screening is performed toidentify modulators that alter a biological function of the expressionproduct of a differentially expressed gene. Again, having identified theimportance of a gene in a particular state, screens are performed toidentify agents that bind and/or modulate the biological activity of thegene product.

In addition, screens are done for genes that are induced in response toa candidate agent. After identifying a modulator (one that suppresses acancer expression pattern leading to a normal expression pattern, or amodulator of a cancer gene that leads to expression of the gene as innormal tissue) a screen is performed to identify genes that arespecifically modulated in response to the agent. Comparing expressionprofiles between normal tissue and agent-treated cancer tissue revealsgenes that are not expressed in normal tissue or cancer tissue, but areexpressed in agent treated tissue, and vice versa. These agent-specificsequences are identified and used by methods described herein for cancergenes or proteins. In particular these sequences and the proteins theyencode are used in marking or identifying agent-treated cells. Inaddition, antibodies are raised against the agent-induced proteins andused to target novel therapeutics to the treated cancer tissue sample.

Modulator-Related Identification and Screening Assays:

Gene Expression-Related Assays

Proteins, nucleic acids, and antibodies of the invention are used inscreening assays. The cancer-associated proteins, antibodies, nucleicacids, modified proteins and cells containing these sequences are usedin screening assays, such as evaluating the effect of drug candidates ona “gene expression profile,” expression profile of polypeptides oralteration of biological function. In one embodiment, the expressionprofiles are used, preferably in conjunction with high throughputscreening techniques to allow monitoring for expression profile genesafter treatment with a candidate agent (e.g., Davis, G F, et al, J BiolScreen 7:69 (2002); Zlokamik, et al., Science 279:84-8 (1998); Heid,Genome Res 6:986-94, 1996).

The cancer proteins, antibodies, nucleic acids, modified proteins andcells containing the native or modified cancer proteins or genes areused in screening assays. That is, the present invention comprisesmethods for screening for compositions which modulate the cancerphenotype or a physiological function of a cancer protein of theinvention. This is done on a gene itself or by evaluating the effect ofdrug candidates on a “gene expression profile” or biological function.In one embodiment, expression profiles are used, preferably inconjunction with high throughput screening techniques to allowmonitoring after treatment with a candidate agent, see Zlokamik, supra.

A variety of assays are executed directed to the genes and proteins ofthe invention. Assays are run on an individual nucleic acid or proteinlevel. That is, having identified a particular gene as up regulated incancer, test compounds are screened for the ability to modulate geneexpression or for binding to the cancer protein of the invention.“Modulation” in this context includes an increase or a decrease in geneexpression. The preferred amount of modulation will depend on theoriginal change of the gene expression in normal versus tissueundergoing cancer, with changes of at least 10%, preferably 50%, morepreferably 100-300%, and in some embodiments 300-1000% or greater. Thus,if a gene exhibits a 4-fold increase in cancer tissue compared to normaltissue, a decrease of about four-fold is often desired; similarly, a10-fold decrease in cancer tissue compared to normal tissue a targetvalue of a 10-fold increase in expression by the test compound is oftendesired. Modulators that exacerbate the type of gene expression seen incancer are also useful, e.g., as an upregulated target in furtheranalyses.

The amount of gene expression is monitored using nucleic acid probes andthe quantification of gene expression levels, or, alternatively, a geneproduct itself is monitored, e.g., through the use of antibodies to thecancer protein and standard immunoassays. Proteomics and separationtechniques also allow for quantification of expression.

Expression Monitoring to Identify Compounds that Modify Gene Expression

In one embodiment, gene expression monitoring, i.e., an expressionprofile, is monitored simultaneously for a number of entities. Suchprofiles will typically involve one or more of the genes of FIG. 2. Inthis embodiment, e.g., cancer nucleic acid probes are attached tobiochips to detect and quantify cancer sequences in a particular cell.Alternatively, PCR can be used. Thus, a series, e.g., wells of amicrotiter plate, can be used with dispensed primers in desired wells. APCR reaction can then be performed and analyzed for each well.

Expression monitoring is performed to identify compounds that modify theexpression of one or more cancer-associated sequences, e.g., apolynucleotide sequence set out in FIG. 2. Generally, a test modulatoris added to the cells prior to analysis. Moreover, screens are alsoprovided to identify agents that modulate cancer, modulate cancerproteins of the invention, bind to a cancer protein of the invention, orinterfere with the binding of a cancer protein of the invention and anantibody or other binding partner.

In one embodiment, high throughput screening methods involve providing alibrary containing a large number of potential therapeutic compounds(candidate compounds). Such “combinatorial chemical libraries” are thenscreened in one or more assays to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve asconventional lead “compounds,” as compounds for screening, or astherapeutics.

In certain embodiments, combinatorial libraries of potential modulatorsare screened for an ability to bind to a cancer polypeptide or tomodulate activity. Conventionally, new chemical entities with usefulproperties are generated by identifying a chemical compound (called a“lead compound”) with some desirable property or activity, e.g.,inhibiting activity, creating variants of the lead compound, andevaluating the property and activity of those variant compounds. Often,high throughput screening (HTS) methods are employed for such ananalysis.

As noted above, gene expression monitoring is conveniently used to testcandidate modulators (e.g., protein, nucleic acid or small molecule).After the candidate agent has been added and the cells allowed toincubate for a period, the sample containing a target sequence to beanalyzed is, e.g., added to a biochip.

If required, the target sequence is prepared using known techniques. Forexample, a sample is treated to lyse the cells, using known lysisbuffers, electroporation, etc., with purification and/or amplificationsuch as PCR performed as appropriate. For example, an in vitrotranscription with labels covalently attached to the nucleotides isperformed. Generally, the nucleic acids are labeled with biotin-FITC orPE, or with cy3 or cy5.

The target sequence can be labeled with, e.g., a fluorescent, achemiluminescent, a chemical, or a radioactive signal, to provide ameans of detecting the target sequence's specific binding to a probe.The label also can be an enzyme, such as alkaline phosphatase orhorseradish peroxidase, which when provided with an appropriatesubstrate produces a product that is detected. Alternatively, the labelis a labeled compound or small molecule, such as an enzyme inhibitor,that binds but is not catalyzed or altered by the enzyme. The label alsocan be a moiety or compound, such as, an epitope tag or biotin whichspecifically binds to streptavidin. For the example of biotin, thestreptavidin is labeled as described above, thereby, providing adetectable signal for the bound target sequence. Unbound labeledstreptavidin is typically removed prior to analysis.

As will be appreciated by those in the art, these assays can be directhybridization assays or can comprise “sandwich assays”, which includethe use of multiple probes, as is generally outlined in U.S. Pat. Nos.5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670;5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118;5,359,100; 5,124,246; and 5,681,697. In this embodiment, in general, thetarget nucleic acid is prepared as outlined above, and then added to thebiochip comprising a plurality of nucleic acid probes, under conditionsthat allow the formation of a hybridization complex.

A variety of hybridization conditions are used in the present invention,including high, moderate and low stringency conditions as outlinedabove. The assays are generally run under stringency conditions whichallow formation of the label probe hybridization complex only in thepresence of target. Stringency can be controlled by altering a stepparameter that is a thermodynamic variable, including, but not limitedto, temperature, formamide concentration, salt concentration, chaotropicsalt concentration pH, organic solvent concentration, etc. Theseparameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus, it can be desirableto perform certain steps at higher stringency conditions to reducenon-specific binding.

The reactions outlined herein can be accomplished in a variety of ways.Components of the reaction can be added simultaneously, or sequentially,in different orders, with preferred embodiments outlined below. Inaddition, the reaction may include a variety of other reagents. Theseinclude salts, buffers, neutral proteins, e.g. albumin, detergents, etc.which can be used to facilitate optimal hybridization and detection,and/or reduce nonspecific or background interactions. Reagents thatotherwise improve the efficiency of the assay, such as proteaseinhibitors, nuclease inhibitors, anti-microbial agents, etc., may alsobe used as appropriate, depending on the sample preparation methods andpurity of the target. The assay data are analyzed to determine theexpression levels of individual genes, and changes in expression levelsas between states, forming a gene expression profile.

Biological Activity-Related Assays

The invention provides methods identify or screen for a compound thatmodulates the activity of a cancer-related gene or protein of theinvention. The methods comprise adding a test compound, as definedabove, to a cell comprising a cancer protein of the invention. The cellscontain a recombinant nucleic acid that encodes a cancer protein of theinvention. In another embodiment, a library of candidate agents istested on a plurality of cells.

In one aspect, the assays are evaluated in the presence or absence orprevious or subsequent exposure of physiological signals, e.g. hormones,antibodies, peptides, antigens, cytokines, growth factors, actionpotentials, pharmacological agents including chemotherapeutics,radiation, carcinogenics, or other cells (i.e., cell-cell contacts). Inanother example, the determinations are made at different stages of thecell cycle process. In this way, compounds that modulate genes orproteins of the invention are identified. Compounds with pharmacologicalactivity are able to enhance or interfere with the activity of thecancer protein of the invention. Once identified, similar structures areevaluated to identify critical structural features of the compound.

In one embodiment, a method of modulating (e.g., inhibiting) cancer celldivision is provided; the method comprises administration of a cancermodulator. In another embodiment, a method of modulating (e.g.,inhibiting) cancer is provided; the method comprises administration of acancer modulator. In a further embodiment, methods of treating cells orindividuals with cancer are provided; the method comprisesadministration of a cancer modulator.

In one embodiment, a method for modulating the status of a cell thatexpresses a gene of the invention is provided. As used herein statuscomprises such art-accepted parameters such as growth, proliferation,survival, function, apoptosis, senescence, location, enzymatic activity,signal transduction, etc. of a cell. In one embodiment, a cancerinhibitor is an antibody as discussed above. In another embodiment, thecancer inhibitor is an antisense molecule. A variety of cell growth,proliferation, and metastasis assays are known to those of skill in theart, as described herein.

High Throughput Screening to Identify Modulators

The assays to identify suitable modulators are amenable to highthroughput screening. Preferred assays thus detect enhancement orinhibition of cancer gene transcription, inhibition or enhancement ofpolypeptide expression, and inhibition or enhancement of polypeptideactivity.

In one embodiment, modulators evaluated in high throughput screeningmethods are proteins, often naturally occurring proteins or fragments ofnaturally occurring proteins. Thus, e.g., cellular extracts containingproteins, or random or directed digests of proteinaceous cellularextracts, are used. In this way, libraries of proteins are made forscreening in the methods of the invention. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred. Particularly useful test compound will be directedto the class of proteins to which the target belongs, e.g., substratesfor enzymes, or ligands and receptors.

Use of Soft Agar Growth and Colony Formation to Identify andCharacterize Modulators

Normal cells require a solid substrate to attach and grow. When cellsare transformed, they lose this phenotype and grow detached from thesubstrate. For example, transformed cells can grow in stirred suspensionculture or suspended in semi-solid media, such as semi-solid or softagar. The transformed cells, when transfected with tumor suppressorgenes, can regenerate normal phenotype and once again require a solidsubstrate to attach to and grow. Soft agar growth or colony formation inassays are used to identify modulators of cancer sequences, which whenexpressed in host cells, inhibit abnormal cellular proliferation andtransformation. A modulator reduces or eliminates the host cells'ability to grow suspended in solid or semisolid media, such as agar.

Techniques for soft agar growth or colony formation in suspension assaysare described in Freshney, Culture of Animal Cells a Manual of BasicTechnique (3rd ed., 1994). See also, the methods section of Garkavtsevet al. (1996), supra.

Evaluation of Contact Inhibition and Growth Density Limitation toIdentify and Characterize Modulators

Normal cells typically grow in a flat and organized pattern in cellculture until they touch other cells. When the cells touch one another,they are contact inhibited and stop growing. Transformed cells, however,are not contact inhibited and continue to grow to high densities indisorganized foci. Thus, transformed cells grow to a higher saturationdensity than corresponding normal cells. This is detectedmorphologically by the formation of a disoriented monolayer of cells orcells in foci. Alternatively, labeling index with (³H)-thymidine atsaturation density is used to measure density limitation of growth,similarly an MTT or Alamar blue assay will reveal proliferation capacityof cells and the ability of modulators to affect same. See Freshney(1994), supra. Transformed cells, when transfected with tumor suppressorgenes, can regenerate a normal phenotype and become contact inhibitedand would grow to a lower density.

In this assay, labeling index with ³H)-thymidine at saturation densityis a preferred method of measuring density limitation of growth.Transformed host cells are transfected with a cancer-associated sequenceand are grown for 24 hours at saturation density in non-limiting mediumconditions. The percentage of cells labeling with (³H)-thymidine isdetermined by incorporated cpm.

Contact independent growth is used to identify modulators of cancersequences, which had led to abnormal cellular proliferation andtransformation. A modulator reduces or eliminates contact independentgrowth, and returns the cells to a normal phenotype.

Evaluation of Growth Factor or Serum Dependence to Identify andCharacterize Modulators

Transformed cells have lower serum dependence than their normalcounterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966);Eagle et al., J. Exp. Med 131:836-879 (1970)); Freshney, supra. This isin part due to release of various growth factors by the transformedcells. The degree of growth factor or serum dependence of transformedhost cells can be compared with that of control. For example, growthfactor or serum dependence of a cell is monitored in methods to identifyand characterize compounds that modulate cancer-associated sequences ofthe invention.

Use of Tumor-Specific Marker Levels to Identify and CharacterizeModulators

Tumor cells release an increased amount of certain factors (hereinafter“tumor specific markers”) than their normal counterparts. For example,plasminogen activator (PA) is released from human glioma at a higherlevel than from normal brain cells (see, e.g., Gullino, Angiogenesis,Tumor Vascularization, and Potential Interference with Tumor Growth, inBiological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)).Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher levelin tumor cells than their normal counterparts. See, e.g., Folkman,Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF isreleased from endothelial tumors (Ensoli, B et al).

Various techniques which measure the release of these factors aredescribed in Freshney (1994), supra. Also, see, Unkless et al., J. Biol.Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem.251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305 312 (1980);Gullino, Angiogenesis, Tumor Vascularization, and Potential Interferencewith Tumor Growth, in Biological Responses in Cancer, pp. 178-184(Mihich (ed.) 1985); Freshney, Anticancer Res. 5:111-130 (1985). Forexample, tumor specific marker levels are monitored in methods toidentify and characterize compounds that modulate cancer-associatedsequences of the invention.

Invasiveness into Matrigel to Identify and Characterize Modulators

The degree of invasiveness into Matrigel or an extracellular matrixconstituent can be used as an assay to identify and characterizecompounds that modulate cancer associated sequences. Tumor cells exhibita positive correlation between malignancy and invasiveness of cells intoMatrigel or some other extracellular matrix constituent. In this assay,tumorigenic cells are typically used as host cells. Expression of atumor suppressor gene in these host cells would decrease invasiveness ofthe host cells. Techniques described in Cancer Res. 1999; 59:6010;Freshney (1994), supra, can be used. Briefly, the level of invasion ofhost cells is measured by using filters coated with Matrigel or someother extracellular matrix constituent. Penetration into the gel, orthrough to the distal side of the filter, is rated as invasiveness, andrated histologically by number of cells and distance moved, or byprelabeling the cells with ¹²⁵1 and counting the radioactivity on thedistal side of the filter or bottom of the dish. See, e.g., Freshney(1984), supra.

Evaluation of Tumor Growth In Vivo to Identify and CharacterizeModulators

Effects of cancer-associated sequences on cell growth are tested intransgenic or immune-suppressed organisms. Transgenic organisms areprepared in a variety of art-accepted ways. For example, knock-outtransgenic organisms, e.g., mammals such as mice, are made, in which acancer gene is disrupted or in which a cancer gene is inserted.Knock-out transgenic mice are made by insertion of a marker gene orother heterologous gene into the endogenous cancer gene site in themouse genome via homologous recombination. Such mice can also be made bysubstituting the endogenous cancer gene with a mutated version of thecancer gene, or by mutating the endogenous cancer gene, e.g., byexposure to carcinogens.

To prepare transgenic chimeric animals, e.g., mice, a DNA construct isintroduced into the nuclei of embryonic stem cells. Cells containing thenewly engineered genetic lesion are injected into a host mouse embryo,which is re-implanted into a recipient female. Some of these embryosdevelop into chimeric mice that possess germ cells some of which arederived from the mutant cell line. Therefore, by breeding the chimericmice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric mice can be derived according to U.S. Pat. No.6,365,797, issued 2 Apr. 2002; U.S. Pat. No. 6,107,540 issued 22 Aug.2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual,Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and EmbryonicStem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington,D.C., (1987).

Alternatively, various immune-suppressed or immune-deficient hostanimals can be used. For example, a genetically athymic “nude” mouse(see, e.g., Giovanella et al., J. Nat Cancer Inst. 52:921 (1974)), aSCID mouse, a thymectomized mouse, or an irradiated mouse (see, e.g.,Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer41:52 (1980)) can be used as a host. Transplantable tumor cells(typically about 10⁶ cells) injected into isogenic hosts produceinvasive tumors in a high proportion of cases, while normal cells ofsimilar origin will not. In hosts which developed invasive tumors, cellsexpressing cancer-associated sequences are injected subcutaneously ororthotopically. Mice are then separated into groups, including controlgroups and treated experimental groups) e.g. treated with a modulator).After a suitable length of time, preferably 4-8 weeks, tumor growth ismeasured (e.g., by volume or by its two largest dimensions, or weight)and compared to the control. Tumors that have statistically significantreduction (using, e.g., Student's T test) are said to have inhibitedgrowth.

In Vitro Assays to Identify and Characterize Modulators

Assays to identify compounds with modulating activity can be performedin vitro. For example, a cancer polypeptide is first contacted with apotential modulator and incubated for a suitable amount of time, e.g.,from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levelsare determined in vitro by measuring the level of protein or mRNA. Thelevel of protein is measured using immunoassays such as Westernblotting, ELISA and the like with an antibody that selectively binds tothe cancer polypeptide or a fragment thereof. For measurement of mRNA,amplification, e.g., using PCR, LCR, or hybridization assays, e. g.,Northern hybridization, RNAse protection, dot blotting, are preferred.The level of protein or mRNA is detected using directly or indirectlylabeled detection agents, e.g., fluorescently or radioactively labelednucleic acids, radioactively or enzymatically labeled antibodies, andthe like, as described herein.

Alternatively, a reporter gene system can be devised using a cancerprotein promoter operably linked to a reporter gene such as luciferase,green fluorescent protein, CAT, or P-gal. The reporter construct istypically transfected into a cell. After treatment with a potentialmodulator, the amount of reporter gene transcription, translation, oractivity is measured according to standard techniques known to those ofskill in the art (Davis G F, supra; Gonzalez, J. & Negulescu, P. Curr.Opin. Biotechnol. 1998: 9:624).

As outlined above, in vitro screens are done on individual genes andgene products. That is, having identified a particular differentiallyexpressed gene as important in a particular state, screening ofmodulators of the expression of the gene or the gene product itself isperformed.

In one embodiment, screening for modulators of expression of specificgene(s) is performed. Typically, the expression of only one or a fewgenes is evaluated. In another embodiment, screens are designed to firstfind compounds that bind to differentially expressed proteins. Thesecompounds are then evaluated for the ability to modulate differentiallyexpressed activity. Moreover, once initial candidate compounds areidentified, variants can be further screened to better evaluatestructure activity relationships.

Binding Assays to Identify and Characterize Modulators

In binding assays in accordance with the invention, a purified orisolated gene product of the invention is generally used. For example,antibodies are generated to a protein of the invention, and immunoassaysare run to determine the amount and/or location of protein.Alternatively, cells comprising the cancer proteins are used in theassays.

Thus, the methods comprise combining a cancer protein of the inventionand a candidate compound such as a ligand, and determining the bindingof the compound to the cancer protein of the invention. Preferredembodiments utilize the human cancer protein; animal models of humandisease of can also be developed and used. Also, other analogousmammalian proteins also can be used as appreciated by those of skill inthe art. Moreover, in some embodiments variant or derivative cancerproteins are used.

Generally, the cancer protein of the invention, or the ligand, isnon-diffusibly bound to an insoluble support. The support can, e.g., beone having isolated sample receiving areas (a microtiter plate, anarray, etc.). The insoluble supports can be made of any composition towhich the compositions can be bound, is readily separated from solublematerial, and is otherwise compatible with the overall method ofscreening. The surface of such supports can be solid or porous and ofany convenient shape.

Examples of suitable insoluble supports include microtiter plates,arrays, membranes and beads. These are typically made of glass, plastic(e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon™,etc. Microtiter plates and arrays are especially convenient because alarge number of assays can be carried out simultaneously, using smallamounts of reagents and samples. The particular manner of binding of thecomposition to the support is not crucial so long as it is compatiblewith the reagents and overall methods of the invention, maintains theactivity of the composition and is nondiffusable. Preferred methods ofbinding include the use of antibodies which do not sterically blockeither the ligand binding site or activation sequence when attaching theprotein to the support, direct binding to “sticky” or ionic supports,chemical crosslinking, the synthesis of the protein or agent on thesurface, etc. Following binding of the protein or ligand/binding agentto the support, excess unbound material is removed by washing. Thesample receiving areas may then be blocked through incubation withbovine serum albumin (BSA), casein or other innocuous protein or othermoiety.

Once a cancer protein of the invention is bound to the support, and atest compound is added to the assay. Alternatively, the candidatebinding agent is bound to the support and the cancer protein of theinvention is then added. Binding agents include specific antibodies,non-natural binding agents identified in screens of chemical libraries,peptide analogs, etc.

Of particular interest are assays to identify agents that have a lowtoxicity for human cells. A wide variety of assays can be used for thispurpose, including proliferation assays, cAMP assays, labeled in vitroprotein-protein binding assays, electrophoretic mobility shift assays,immunoassays for protein binding, functional assays (phosphorylationassays, etc.) and the like.

A determination of binding of the test compound (ligand, binding agent,modulator, etc.) to a cancer protein of the invention can be done in anumber of ways. The test compound can be labeled, and binding determineddirectly, e.g., by attaching all or a portion of the cancer protein ofthe invention to a solid support, adding a labeled candidate compound(e.g., a fluorescent label), washing off excess reagent, and determiningwhether the label is present on the solid support. Various blocking andwashing steps can be utilized as appropriate.

In certain embodiments, only one of the components is labeled, e.g., aprotein of the invention or ligands labeled. Alternatively, more thanone component is labeled with different labels, e.g., I¹²⁵, for theproteins and a fluorophor for the compound. Proximity reagents, e.g.,quenching or energy transfer reagents are also useful.

Competitive Binding to Identify and Characterize Modulators

In one embodiment, the binding of the “test compound” is determined bycompetitive binding assay with a “competitor.” The competitor is abinding moiety that binds to the target molecule (e.g., a cancer proteinof the invention). Competitors include compounds such as antibodies,peptides, binding partners, ligands, etc. Under certain circumstances,the competitive binding between the test compound and the competitordisplaces the test compound. In one embodiment, the test compound islabeled. Either the test compound, the competitor, or both, is added tothe protein for a time sufficient to allow binding. Incubations areperformed at a temperature that facilitates optimal activity, typicallybetween four and 40° C. Incubation periods are typically optimized,e.g., to facilitate rapid high throughput screening; typically betweenzero and one hour will be sufficient. Excess reagent is generallyremoved or washed away. The second component is then added, and thepresence or absence of the labeled component is followed, to indicatebinding.

In one embodiment, the competitor is added first, followed by the testcompound. Displacement of the competitor is an indication that the testcompound is binding to the cancer protein and thus is capable of bindingto, and potentially modulating, the activity of the cancer protein. Inthis embodiment, either component can be labeled. Thus, e.g., if thecompetitor is labeled, the presence of label in the post-test compoundwash solution indicates displacement by the test compound.Alternatively, if the test compound is labeled, the presence of thelabel on the support indicates displacement.

In an alternative embodiment, the test compound is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor indicates that the test compound binds to thecancer protein with higher affinity than the competitor. Thus, if thetest compound is labeled, the presence of the label on the support,coupled with a lack of competitor binding, indicates that the testcompound binds to and thus potentially modulates the cancer protein ofthe invention.

Accordingly, the competitive binding methods comprise differentialscreening to identity agents that are capable of modulating the activityof the cancer proteins of the invention. In this embodiment, the methodscomprise combining a cancer protein and a competitor in a first sample.A second sample comprises a test compound, the cancer protein, and acompetitor. The binding of the competitor is determined for bothsamples, and a change, or difference in binding between the two samplesindicates the presence of an agent capable of binding to the cancerprotein and potentially modulating its activity. That is, if the bindingof the competitor is different in the second sample relative to thefirst sample, the agent is capable of binding to the cancer protein.

Alternatively, differential screening is used to identify drugcandidates that bind to the native cancer protein, but cannot bind tomodified cancer proteins. For example the structure of the cancerprotein is modeled and used in rational drug design to synthesize agentsthat interact with that site, agents which generally do not bind tosite-modified proteins. Moreover, such drug candidates that affect theactivity of a native cancer protein are also identified by screeningdrugs for the ability to either enhance or reduce the activity of suchproteins.

Positive controls and negative controls can be used in the assays.Preferably control and test samples are performed in at least triplicateto obtain statistically significant results. Incubation of all samplesoccurs for a time sufficient to allow for the binding of the agent tothe protein. Following incubation, samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples can be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents can be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc. which are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,can be used. The mixture of components is added in an order thatprovides for the requisite binding.

Use of Polynucleotides to Down-Regulate or Inhibit a Protein of theInvention.

Polynucleotide modulators of cancer can be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand-binding molecule, as described in WO 91/04753. Suitableligand-binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell. Alternatively, a polynucleotide modulator ofcancer can be introduced into a cell containing the target nucleic acidsequence, e.g., by formation of a polynucleotide-lipid complex, asdescribed in WO 90/10448. It is understood that the use of antisensemolecules or knock out and knock in models may also be used in screeningassays as discussed above, in addition to methods of treatment.

Inhibitory and Antisense Nucleotides

In certain embodiments, the activity of a cancer-associated protein isdown-regulated, or entirely inhibited, by the use of antisensepolynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleicacid complementary to, and which can preferably hybridize specificallyto, a coding mRNA nucleic acid sequence, e.g., a cancer protein of theinvention, mRNA, or a subsequence thereof. Binding of the antisensepolynucleotide to the mRNA reduces the translation and/or stability ofthe mRNA.

In the context of this invention, antisense polynucleotides can comprisenaturally occurring nucleotides, or synthetic species formed fromnaturally occurring subunits or their close homologs. Antisensepolynucleotides may also have altered sugar moieties or inter-sugarlinkages. Exemplary among these are the phosphorothioate and othersulfur containing species which are known for use in the art. Analogsare comprised by this invention so long as they function effectively tohybridize with nucleotides of the invention. See, e.g., IsisPharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.

Such antisense polynucleotides can readily be synthesized usingrecombinant means, or can be synthesized in vitro. Equipment for suchsynthesis is sold by several vendors, including Applied Biosystems. Thepreparation of other oligonucleotides such as phosphorothioates andalkylated derivatives is also well known to those of skill in the art.

Antisense molecules as used herein include antisense or senseoligonucleotides. Sense oligonucleotides can, e.g., be employed to blocktranscription by binding to the anti-sense strand. The antisense andsense oligonucleotide comprise a single stranded nucleic acid sequence(either RNA or DNA) capable of binding to target mRNA (sense) or DNA(antisense) sequences for cancer molecules. Antisense or senseoligonucleotides, according to the present invention, comprise afragment generally at least about 12 nucleotides, preferably from about12 to 30 nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, e.g., Stein &Cohen (Cancer Res. 48:2659 (1988 and van derKrol et al. (BioTechniques 6:958 (1988)).

Ribozymes

In addition to antisense polynucleotides, ribozymes can be used totarget and inhibit transcription of cancer-associated nucleotidesequences. A ribozyme is an RNA molecule that catalytically cleavesother RNA molecules. Different kinds of ribozymes have been described,including group I ribozymes, hammerhead ribozymes, hairpin ribozymes,RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. inPharmacology 25: 289-317 (1994) for a general review of the propertiesof different ribozymes).

The general features of hairpin ribozymes are described, e.g., in Hampelet al., Nucl. Acids Res. 18:299-304 (1990); European Patent PublicationNo. 0360257; U.S. Pat. No. 5,254,678. Methods of preparing are wellknown to those of skill in the art (see, e.g., WO 94/26877; Ojwang etal., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al.,Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Nati. Acad Sci.USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5: 1151-120(1994); and Yamada et al., Virology 205: 121-126 (1994)).

Use of Modulators in Phenotypic Screening

In one embodiment, a test compound is administered to a population ofcancer cells, which have an associated cancer expression profile. By“administration” or “contacting” herein is meant that the modulator isadded to the cells in such a manner as to allow the modulator to actupon the cell, whether by uptake and intracellular action, or by actionat the cell surface. In some embodiments, a nucleic acid encoding aproteinaceous agent (i.e., a peptide) is put into a viral construct suchas an adenoviral or retroviral construct, and added to the cell, suchthat expression of the peptide agent is accomplished, e.g., PCTUS97/01019. Regulatable gene therapy systems can also be used. Once themodulator has been administered to the cells, the cells are washed ifdesired and are allowed to incubate under preferably physiologicalconditions for some period. The cells are then harvested and a new geneexpression profile is generated. Thus, e.g., cancer tissue is screenedfor agents that modulate, e.g., induce or suppress, the cancerphenotype. A change in at least one gene, preferably many, of theexpression profile indicates that the agent has an effect on canceractivity. Similarly, altering a biological function or a signalingpathway is indicative of modulator activity. By defining such asignature for the cancer phenotype, screens for new drugs that alter thephenotype are devised. With this approach, the drug target need not beknown and need not be represented in the original gene/proteinexpression screening platform, nor does the level of transcript for thetarget protein need to change. The modulator inhibiting function willserve as a surrogate marker.

As outlined above, screens are done to assess genes or gene products.That is, having identified a particular differentially expressed gene asimportant in a particular state, screening of modulators of either theexpression of the gene or the gene product itself is performed.

Use of Modulators to Affect Peptides of the Invention

Measurements of cancer polypeptide activity, or of the cancer phenotypeare performed using a variety of assays. For example, the effects ofmodulators upon the function of a cancer polypeptide(s) are measured byexamining parameters described above. A physiological change thataffects activity is used to assess the influence of a test compound onthe polypeptides of this invention. When the functional outcomes aredetermined using intact cells or animals, a variety of effects can beassesses such as, in the case of a cancer associated with solid tumors,tumor growth, tumor metastasis, neovascularization, hormone release,transcriptional changes to both known and uncharacterized geneticmarkers (e.g., by Northern blots), changes in cell metabolism such ascell growth or pH changes, and changes in intracellular secondmessengers such as cGNIP.

Methods of Identifying Characterizing Cancer-Associated Sequences

Expression of various gene sequences is correlated with cancer.Accordingly, disorders based on mutant or variant cancer genes aredetermined. In one embodiment, the invention provides methods foridentifying cells containing variant cancer genes, e.g., determining thepresence of, all or part, the sequence of at least one endogenous cancergene in a cell. This is accomplished using any number of sequencingtechniques. The invention comprises methods of identifying the cancergenotype of an individual, e.g., determining all or part of the sequenceof at least one gene of the invention in the individual. This isgenerally done in at least one tissue of the individual, e.g., a tissueset forth in Table I, and may include the evaluation of a number oftissues or different samples of the same tissue. The method may includecomparing the sequence of the sequenced gene to a known cancer gene,i.e., a wild-type gene to determine the presence of family members,homologies, mutations or variants. The sequence of all or part of thegene can then be compared to the sequence of a known cancer gene todetermine if any differences exist. This is done using any number ofknown homology programs, such as BLAST, Bestfit, etc. The presence of adifference in the sequence between the cancer gene of the patient andthe known cancer gene correlates with a disease state or a propensityfor a disease state, as outlined herein.

In a preferred embodiment, the cancer genes are used as probes todetermine the number of copies of the cancer gene in the genome. Thecancer genes are used as probes to determine the chromosomallocalization of the cancer genes. Information such as chromosomallocalization finds use in providing a diagnosis or prognosis inparticular when chromosomal abnormalities such as translocations, andthe like are identified in the cancer gene locus.

XIV.) RNAI AND THERAPEUTIC USE OF SMALL INTERFERING RNA (SIRNAS)

The present invention is also directed towards siRNA oligonucleotides,particularly double stranded RNAs encompassing at least a fragment ofthe STEAP-1 coding region or 5″ UTR regions, or complement, or anyantisense oligonucleotide specific to the STEAP-1 sequence. In oneembodiment such oligonucleotides are used to elucidate a function ofSTEAP-1, or are used to screen for or evaluate modulators of STEAP-1function or expression. In another embodiment, gene expression ofSTEAP-1 is reduced by using siRNA transfection and results insignificantly diminished proliferative capacity of transformed cancercells that endogenously express the antigen; cells treated with specificSTEAP-1 siRNAs show reduced survival as measured, e.g., by a metabolicreadout of cell viability, correlating to the reduced proliferativecapacity. Thus, STEAP-1 siRNA compositions comprise siRNA (doublestranded RNA) that correspond to the nucleic acid ORF sequence of theSTEAP-1 protein or subsequences thereof; these subsequences aregenerally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more than 35contiguous RNA nucleotides in length and contain sequences that arecomplementary and non-complementary to at least a portion of the mRNAcoding sequence In a preferred embodiment, the subsequences are 19-25nucleotides in length, most preferably 21-23 nucleotides in length.

RNA interference is a novel approach to silencing genes in vitro and invivo, thus small double stranded RNAs (siRNAs) are valuable therapeuticagents. The power of siRNAs to silence specific gene activities has nowbeen brought to animal models of disease and is used in humans as well.For example, hydrodynamic infusion of a solution of siRNA into a mousewith a siRNA against a particular target has been proven to betherapeutically effective.

The pioneering work by Song et al. indicates that one type of entirelynatural nucleic acid, small interfering RNAs (siRNAs), served astherapeutic agents even without further chemical modification (Song, E.,et al. “RNA interference targeting Fas protects mice from fulminanthepatitis” Nat. Med. 9(3): 347-51(2003)). This work provided the firstin vivo evidence that infusion of siRNAs into an animal could alleviatedisease. In that case, the authors gave mice injections of siRNAdesigned to silence the FAS protein (a cell death receptor that whenover-activated during inflammatory response induces hepatocytes andother cells to die). The next day, the animals were given an antibodyspecific to Fas. Control mice died of acute liver failure within a fewdays, while over 80% of the siRNA-treated mice remained free fromserious disease and survived. About 80% to 90% of their liver cellsincorporated the naked siRNA oligonucleotides. Furthermore, the RNAmolecules functioned for 10 days before losing effect after 3 weeks.

For use in human therapy, siRNA is delivered by efficient systems thatinduce long-lasting RNAi activity. A major caveat for clinical use isdelivering siRNAs to the appropriate cells. Hepatocytes seem to beparticularly receptive to exogenous RNA. Today, targets located in theliver are attractive because liver is an organ that can be readilytargeted by nucleic acid molecules and viral vectors. However, othertissue and organs targets are preferred as well.

Formulations of siRNAs with compounds that promote transit across cellmembranes are used to improve administration of siRNAs in therapy.Chemically modified synthetic siRNA, that are resistant to nucleases andhave serum stability have concomitant enhanced duration of RNAi effects,are an additional embodiment.

Thus, siRNA technology is a therapeutic for human malignancy by deliveryof siRNA molecules directed to STEAP-1 to individuals with the cancers,such as those listed in Table 1. Such administration of siRNAs leads toreduced growth of cancer cells expressing STEAP-1, and provides ananti-tumor therapy, lessening the morbidity and/or mortality associatedwith malignancy.

The effectiveness of this modality of gene product knockdown issignificant when measured in vitro or in vivo. Effectiveness in vitro isreadily demonstrable through application of siRNAs to cells in culture(as described above) or to aliquots of cancer patient biopsies when invitro methods are used to detect the reduced expression of STEAP-1protein.

XV.) KITS/ARTICLES OF MANUFACTURE

For use in the laboratory, prognostic, prophylactic, diagnostic andtherapeutic applications described herein, kits are within the scope ofthe invention. Such kits can comprise a carrier, package, or containerthat is compartmentalized to receive one or more containers such asvials, tubes, and the like, each of the container(s) comprising one ofthe separate elements to be used in the method, along with a label orinsert comprising instructions for use, such as a use described herein.For example, the container(s) can comprise a probe that is or can bedetectably labeled. Such probe can be an antibody or polynucleotidespecific for a protein or a gene or message of the invention,respectively. Where the method utilizes nucleic acid hybridization todetect the target nucleic acid, the kit can also have containerscontaining nucleotide(s) for amplification of the target nucleic acidsequence. Kits can comprise a container comprising a reporter, such as abiotin-binding protein, such as avidin or streptavidin, bound to areporter molecule, such as an enzymatic, fluorescent, or radioisotopelabel; such a reporter can be used with, e.g., a nucleic acid orantibody. The kit can include all or part of the amino acid sequences inFIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecule thatencodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers associated therewith thatcomprise materials desirable from a commercial and user standpoint,including buffers, diluents, filters, needles, syringes; carrier,package, container, vial and/or tube labels listing contents and/orinstructions for use, and package inserts with instructions for use.

A label can be present on or with the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, such as a prognostic, prophylactic, diagnostic orlaboratory application, and can also indicate directions for either invivo or in vitro use, such as those described herein. Directions and orother information can also be included on an insert(s) or label(s) whichis included with or on the kit. The label can be on or associated withthe container. A label a can be on a container when letters, numbers orother characters forming the label are molded or etched into thecontainer itself; a label can be associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. The label can indicate that the compositionis used for diagnosing, treating, prophylaxing or prognosing acondition, such as a neoplasia of a tissue set forth in Table I.

The terms “kit” and “article of manufacture” can be used as synonyms.

In another embodiment of the invention, an article(s) of manufacturecontaining compositions, such as amino acid sequence(s), smallmolecule(s), nucleic acid sequence(s), and/or antibody(s), e.g.,materials useful for the diagnosis, prognosis, prophylaxis and/ortreatment of neoplasias of tissues such as those set forth in Table I isprovided. The article of manufacture typically comprises at least onecontainer and at least one label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers can beformed from a variety of materials such as glass, metal or plastic. Thecontainer can hold amino acid sequence(s), small molecule(s), nucleicacid sequence(s), cell population(s) and/or antibody(s). In oneembodiment, the container holds a polynucleotide for use in examiningthe mRNA expression profile of a cell, together with reagents used forthis purpose. In another embodiment a container comprises an antibody,binding fragment thereof or specific binding protein for use inevaluating protein expression of STEAP-1 in cells and tissues, or forrelevant laboratory, prognostic, diagnostic, prophylactic andtherapeutic purposes; indications and/or directions for such uses can beincluded on or with such container, as can reagents and othercompositions or tools used for these purposes. In another embodiment, acontainer comprises materials for eliciting a cellular or humoral immuneresponse, together with associated indications and/or directions. Inanother embodiment, a container comprises materials for adoptiveimmunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL),together with associated indications and/or directions; reagents andother compositions or tools used for such purpose can also be included.

The container can alternatively hold a composition that is effective fortreating, diagnosis, prognosing or prophylaxing a condition and can havea sterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agents in the composition can be anantibody capable of specifically binding STEAP-1 and modulating thefunction of STEAP-1.

The article of manufacture can further comprise a second containercomprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and/or dextrose solution.It can further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, stirrers,needles, syringes, and/or package inserts with indications and/orinstructions for use.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which is intended tolimit the scope of the invention.

Example 1 SSH-Generated Isolation of cDNA Fragment of the STEAP-1 GeneMaterials and Methods LAPC Xenoarafts:

LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) andgenerated as described (Klein et al, 1997, Nature Med. 3: 402-408; Craftet al., 1999, Cancer Res. 59: 5030-5036). Androgen dependent andindependent LAPC-4 xenografts (LAPC-4 AD and Al, respectively) andLAPC-9 xenografts (LAPC-9 AD and Al, respectively) were grown in intactmale SCID mice or in castrated males, respectively, and were passaged assmall tissue chunks in recipient males. LAPC-4 Al xenografts werederived from LAPC-4 AD tumors and LAPC-9 Al xenografts were derived fromLAPC-9 AD tumors. To generate the Al xenografts, male mice bearing LAPCAD tumors were castrated and maintained for 2-3 months. After the LAPCtumors re-grew, the tumors were harvested and passaged in castratedmales or in female SCID mice.

LAPC-4 AD xenografts were grown intratibially as follows. LAPC-4 ADxenograft tumor tissue grown subcutaneously was minced into 1-2 mm³sections while the tissue was bathed in 1× Iscoves medium, minced tissuewas then centrifuged at 1.3K rpm for 4 minutes, the supernatant wasresuspended in 10 ml ice cold 1× Iscoves medium and centrifuged at 1.3Krpm for 4 minutes. The pellet was then resuspended in 1× Iscoves with 1%pronase E and incubated for 20 minutes at room temperature with mildrocking agitation followed by incubation on ice for 2-4 minutes.Filtrate was centrifuged at 1.3K rpm for 4 minutes, and the pronase wasremoved from the aspirated pellet by resuspending in 10 ml Iscoves andre-centrifuging. Clumps of cells were then plated in PrEGM medium andgrown overnight. The cells were then harvested, filtered, washed 2×RPMI,and counted. Approximately 50,000 cells were mixed with and equal volumeof ice-cold Matrigel on ice, and surgically injected into the proximaltibial metaphyses of SCID mice via a 27 gauge needle. After 10-12 weeks,LAPC-4 tumors growing in bone marrow were recovered.

Cell Lines and Tissues:

Human cell lines (e.g., HeLa) were obtained from the ATCC and weremaintained in DMEM with 5% fetal calf serum. Human tissues for RNA andprotein analyses were obtained from the Human Tissue Resource Center(HTRC) at the UCLA (Los Angeles, Calif.) and from QualTek, Inc. (SantaBarbara, Calif.).

RNA Isolation:

Tumor tissue and cell lines were homogenized in Trizol reagent (LifeTechnologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer): (SEQ ID NO: 68) 5'TTTTGATCAAGCTT₃₀3'Adaptor 1: (SEQ ID NO: 69)5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO: 70)3'GGCCCGTCCTAG5' Adaptor 2: (SEQ ID NO: 71)5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 72)3'CGGCTCCTAG5' PCR primer 1: (SEQ ID NO: 73) 5'CTAATACGACTCACTATAGGGC3′Nested primer (NP)1: (SEQ ID NO: 74) 5'TCGAGCGGCCGCCCGGGCAGGA3′Nested primer (NP)2: (SEQ ID NO: 75) 5'AGCGTGGTCGCGGCCGAGGA3'

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes, which may be up-regulated in androgen dependentprostate cancer compared to benign prostatic hyperplasia (BPH).

Double stranded cDNAs corresponding to the LAPC-4 AD xenograft (tester)and the BPH tissue (driver) were synthesized from 2 μg of poly(A)+RNAisolated from xenograft and BPH tissue, as described above, usingCLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotideRSACDN as primer. First- and second-strand synthesis were carried out asdescribed in the Kit's user manual protocol (CLONTECH Protocol No.PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with RsaI for 3 hrs. at 37° C. Digested cDNA was extracted withpheno/chloroforrn (1:1) and ethanol precipitated.

Driver cDNA (BPH) was generated by combining in a 4 to 1 ratio Rsa Idigested BPH cDNA with digested cDNA from mouse liver, in order toensure that murine genes were subtracted from the tester cDNA (LAPC-4AD).

Tester cDNA (LAPC-4 AD) was generated by diluting 1 μl of Rsa I digestedLAPC-4 AD cDNA (400 ng) in 5 μl of water. The diluted cDNA (2 μl, 160ng) was then ligated to 2 μl of adaptor 1 and adaptor 2 (10 μM), inseparate ligation reactions, in a total volume of 100 at 16° C.overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation wasterminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) adaptor 1-and adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlayed with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification. Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, 72° C. for 1.5 minutes. The PCR products wereanalyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dbEST, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs were generated from 1 μg of mRNA with oligo (dT)12-18priming using the Gibco-BRL Superscript Preamplification system. Themanufacturers protocol was used and included an incubation for 50 min at42° C. with reverse transcriptase followed by RNase H treatment at 37°C. for 20 min. After completing the reaction, the volume was increasedto 200 μl with water prior to normalization. First strand cDNAs from 16different normal human tissues were obtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′ atatcgccgcgctcgtcgtcgacaa3′ (SEQ IDNO: 76) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 77) to amplifyβ-actin. First strand cDNA (5 μl) was amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction was removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: initial denaturation was at 94° C. for 15 sec, followed by a18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 by β-actinbands from multiple tissues were compared by visual inspection. Dilutionfactors for the first strand cDNAs were calculated to result in equalβ-actin band intensities in all tissues after 22 cycles of PCR. Threerounds of normalization were required to achieve equal band intensitiesin all tissues after 22 cycles of PCR.

To determine expression levels of the STEAP-1 gene, 5 μl of normalizedfirst strand cDNA was analyzed by PCR using 25, 30, and 35 cycles ofamplification using the following primer pairs:

(SEQ ID NO: 78) 5′ACT TTG TTG ATG ACC AGG ATI GGA 3′ (SEQ ID NO: 79)5′CAG AAC TTC AGC ACA CAC AGG AAC 3′Semi quantitative expression analysis was achieved by comparing the PCRproducts at cycle numbers that give light band intensities.

Results

Several SSH experiments were conduced as described in the Materials andMethods, supra, and led to the isolation of numerous candidate genefragment clones. All candidate clones were sequenced and subjected tohomology analysis against all sequences in the major public gene and ESTdatabases in order to provide information on the identity of thecorresponding gene and to help guide the decision to analyze aparticular gene for differential expression. In general, gene fragmentswhich had no homology to any known sequence in any of the searcheddatabases, and thus considered to represent novel genes, as well as genefragments showing homology to previously sequenced expressed sequencetags (ESTs), were subjected to differential expression analysis byRT-PCR and/or Northern analysis.

One of the cDNA clones, designated STEAP-1, was 436 bp in length andshowed homology to an EST sequence in the NCI-CGAP tumor gene database.The full length cDNA encoding the STEAP-1 gene was subsequently isolatedusing this cDNA and re-named STEAP-1. The STEAP-1 cDNA nucleotidesequence corresponds to nucleotide residues 150 through 585 in theSTEAP-1 cDNA sequence as shown in FIG. 1. Another clone, designated28P3E1, 561 bp in length showed homology to a number of EST sequences inthe NCI-CGAP tumor gene database or in other databases. Part of the28P3E1 sequence (356 bp) is identical to an EST derived from human fetaltissue. After the full-length STEAP-1 cDNA was obtained and sequenced,it became apparent that this clone also corresponds to STEAP-1 (morespecifically, to residues 622 through the 3′ end of the STEAP-1nucleotide sequence as shown in FIG. 1).

Example 2 Isolation of Full Length STEAP-1 Encoding cDNA

The 436 by STEAP-1 gene fragment (See Example Entitled, “SSH-GeneratedIsolation of cDNA Fragment of the STEAP-1 Gene”) was used to isolateadditional cDNAs encoding the 8P1D4/STEAP-1 gene. Briefly, a normalhuman prostate cDNA library (Clontech) was screened with a labeled probegenerated from the 436 by STEAP-1 cDNA. One of the positive clones,clone 10, is 1195 bp in length and encodes a 339 amino acid proteinhaving nucleotide and encoded amino acid sequences bearing nosignificant homology to any known human genes or proteins (homology to arat Kidney Injury Protein described in International ApplicationWO98/53071). The encoded protein contains at least 6 predictedtransmembrane motifs implying a cell surface orientation Thesestructural features led to the designation “STEAP”, for “SixTransmembrane Epithelial Antigen of the Prostate”.

Subsequent identification of additional “STEAP” proteins led to there-designation of the STEAP-1 gene product as “STEAP-1”. The STEAP-1cDNA and encoded amino acid sequences are shown in FIG. 2A-Q. STEAP-1cDNA clone 10 was deposited with the American Type Culture Collection(“ATCC”) (10801 University Blvd., Manassas, Va. 20110-2209 USA) asplasmid STEAP-1 clone 10.1 on Aug. 26, 1998 as ATCC Accession Number98849. The STEAP-1 cDNA clone can be excised therefrom using EcoRI/Xbaldouble digest (EcoRI at the 5′end, Xbal at the 3′end).

Example 3 Chromosomal Mapping of STEAP-1

Chromosomal localization can implicate genes in disease pathogenesis.Several chromosome mapping approaches are available includingfluorescent in situ hybridization (FISH), human/hamster radiation hybrid(RH) panels (Walter et al., 1994; Nature Genetics 7:22; ResearchGenetics, Huntsville AI), human-rodent somatic cell hybrid panels suchas is available from the Coriell Institute (Camden, N.J.), and genomicviewers utilizing BLAST homologies to sequenced and mapped genomicclones (NCBI, Bethesda, Md.).

STEAP-1 maps to chromosome 7q21 using STEAP-1 sequence and the NCBIBLAST tool: (located on the World Wide Web at(.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlasthtml8AORG=Hs)).

Example 4 Expression Analysis of STEAP-1

Expression of STEAP-1 in stomach cancer patient specimens is shown inFIG. 14( a)-(e). FIG. 14( a) RNA was extracted from normal stomach (N)and from 10 different stomach cancer patient specimens (T). Northernblot with 10 μg of total RNA/lane was probed with STEAP-1 sequence.Results show strong expression of an approximately 1.6 kb STEAP-1 in thestomach tumor tissues. The lower panel represents ethidium bromidestaining of the blot showing quality of the RNA samples.

FIG. 14( b) shows that STEAP-1 was expressed in rectum cancer patienttissues. RNA was extracted from normal rectum (N), rectum cancer patienttumors (T), and rectum cancer metastasis (M). Northern blots with 10 μgof total RNA were probed with the STEAP-1 sequence. Results show strongexpression of STEAP-1 in the rectum cancer patient tissues. The lowerpanel represents ethidium bromide staining of the blot showing qualityof the RNA samples.

Expression of STEAP-1 by RT-PCR demonstrated that STEAP-1 is stronglyexpressed in human umbilical vein endothelial cells (HUVEC) (FIG. 14(c)). First strand cDNA was prepared from HUVEC cells, LAPC-4AD andLAPC-9AD prostate cancer xenografts, as well as from human braintissues. Normalization was performed by PCR using primers to actin andGAPDH. Semi-quantitative PCR, using primers to STEAP-1, was performed at27 and 30 cycles of amplification. As a control, PCR using primers toactin is shown. Results show strong expression of STEAP-1 in HUVEC cellssimilar to the expression detected in prostate cancer xenograft tissues.Expression of STEAP-1 in HUVEC cells indicates that targeting STEAP-1may also target endothelial cells of the neovasculature of the tumors.In FIG. 14( d) picture of the RT-PCR agarose gel is shown. In FIG. 14(e) PCR products were quantitated using the AlphaImager software. Resultsshow strong of expression of STEAP-1 in normal prostate amongst all thenormal tissues tested. Upregulation of STEAP-1 expression was detectedin prostate cancer pool, bladder cancer pool, kidney cancer pool, coloncancer pool, lung cancer pool, ovary cancer pool, and breast cancerpool. Strong expression of STEAP-1 was detected in cancer metastasispool, prostate cancer xenograft pool, and prostate metastasis to lymphnode.

STEAP-1 Expression in lymphoma patient specimens (FIG. 14( f)). Firststrand cDNA was prepared from a panel of lymphoma patient specimens.Normalization was performed by PCR using primers to actin.Semi-quantitative PCR, using primers to STEAP-1, was performed at 26 and30 cycles of amplification. Samples were run on an agarose gel, and PCRproducts were quantitated using the AlphaImager software. Expression wasrecorded as strong or medium, if signal is detected as 26 or 30 cyclesof amplification respectively, and absent if no signal is detected evenat 30 cycles of amplification. Results show expression of STEAP-1 in 8of 11 (72.7%) tumor specimens tested.

Example 5 Splice Variants of STEAP-1

Transcript variants are variants of mature mRNA from the same gene whicharise by alternative transcription or alternative splicing. Alternativetranscripts are transcripts from the same gene but start transcriptionat different points. Splice variants are mRNA variants spliceddifferently from the same transcript. In eukaryotes, when a multi-exongene is transcribed from genomic DNA, the initial RNA is spliced toproduce functional mRNA, which has only exons and is used fortranslation into an amino acid sequence. Accordingly, a given gene canhave zero to many alternative transcripts and each transcript can havezero to many splice variants. Each transcript variant has a unique exonmakeup, and can have different coding and/or non-coding (5′ or 3′ end)portions, from the original transcript. Transcript variants can code forsimilar or different proteins with the same or a similar function or canencode proteins with different functions, and can be expressed in thesame tissue at the same time, or in different tissues at the same time,or in the same tissue at different times, or in different tissues atdifferent times. Proteins encoded by transcript variants can havesimilar or different cellular or extracellular localizations, e.g.,secreted versus intracellular.

Transcript variants are identified by a variety of art-accepted methods.For example, alternative transcripts and splice variants are identifiedby full-length cloning experiment, or by use of full-length transcriptand EST sequences. First, all human ESTs were grouped into clusterswhich show direct or indirect identity with each other. Second, ESTs inthe same cluster were further grouped into sub-clusters and assembledinto a consensus sequence. The original gene sequence is compared to theconsensus sequence(s) or other full-length sequences. Each consensussequence is a potential splice variant for that gene (see, e.g., Kan,Z., et al., Gene structure prediction and alternative splicing analysisusing genomically aligned ESTs, Genome Research, 2001 May,11(5):889-900.) Even when a variant is identified that is not afull-length clone, that portion of the variant is very useful forantigen generation and for further cloning of the full-length splicevariant, using techniques known in the art.

Moreover, computer programs are available in the art that identifytranscript variants based on genomic sequences. Genomic-based transcriptvariant identification programs include FgenesH (A. Salamov and V.Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” GenomeResearch. 2000 April; 10(4):516-22); Grail (URLcompbio.oml.gov/Grail-bin/EmptyGrailForm) and GenScan (URLgenes.mit.edu/GENSCAN.html). For a general discussion of splice variantidentification protocols see., e.g., Southan, C., A genomic perspectiveon human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8; de Souza, S.J., et al., Identification of human chromosome 22 transcribed sequenceswith ORF expressed sequence tags, Proc. Natl. Acad Sci USA. 2000 Nov. 7;97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety oftechniques are available in the art, such as full-length cloning,proteomic validation, PCR-based validation, and 5′ RACE validation, etc.(see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin bankspeninsula: a new termination variant characterized by electrospray massspectrometry, Biochem Biophys Acta. 1999 Aug. 17; 1433(1-2):321-6;Ferranti P, et al., Differential splicing of pre-messenger RNA producesmultiple forms of mature caprine alpha(s1)-casein, Eur J Biochem. 1997Oct. 1; 249(1):1-7. For PCR-based Validation: Wellmann S, et al.,Specific reverse transcription-PCR quantification of vascularendothelial growth factor (VEGF) splice variants by LightCyclertechnology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al.,Discovery of new human beta-defensins using a genomics-based approach,Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACEValidation: Brigle, K. E., et al., Organization of the murine reducedfolate carrier gene and identification of variant splice forms, BiochemBiophys Acta. 1997 Aug. 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers.When the genomic region to which a gene maps is modulated in aparticular cancer, the alternative transcripts or splice variants of thegene are modulated as well. Disclosed herein is that STEAP-1 has aparticular expression profile related to cancer. Alternative transcriptsand splice variants of STEAP-1 may also be involved in cancers in thesame or different tissues, thus serving as tumor-associatedmarkers/antigens.

The exon composition of the original transcript, designated as STEAP-1v.1, is shown in Table LIII. Using the full-length gene and ESTsequences, two transcript variants were identified, designated asSTEAP-1 v.2 and v.3. Compared with STEAP-1 v.1, transcript variantSTEAP-1 v.2 did not splice out intron 4 of STEAP-1 v.1 and variantSTEAP-1 v.3 spliced out one additional exon from intron 4 of STEAP-1v.1, as shown in FIG. 11. Theoretically, each different combination ofexons in spatial order, e.g. exons 2 and 3, is a potential splicevariant. FIG. 11 shows the schematic alignment of exons of thetranscript variants.

Example 6 Single Nucleotide Polymorphisms of STEAP-1

A Single Nucleotide Polymorphism (SNP) is a single base pair variationin a nucleotide sequence at a specific location. At any given point ofthe genome, there are four possible nucleotide base pairs: A/T, C/G, G/Cand T/A. Genotype refers to the specific base pair sequence of one ormore locations in the genome of an individual. Haplotype refers to thebase pair sequence of more than one location on the same DNA molecule(or the same chromosome in higher organisms), often in the context ofone gene or in the context of several tightly linked genes. SNPs thatoccur on a cDNA are called cSNPs. These cSNPs may change amino acids ofthe protein encoded by the gene and thus change the functions of theprotein. Some SNPs cause inherited diseases; others contribute toquantitative variations in phenotype and reactions to environmentalfactors including diet and drugs among individuals. Therefore, SNPsand/or combinations of alleles (called haplotypes) have manyapplications, including diagnosis of inherited diseases, determinationof drug reactions and dosage, identification of genes responsible fordiseases, and analysis of the genetic relationship between individuals(P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect humantraits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M.Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drugreactions,” Trends Pharmacol. Sci. 2001 June; 22(6):298-305; J. H.Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotidepolymorphisms in the isolation of common disease genes,”Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C. Stephensand A. Windemuth, “The predictive power of haplotypes in clinicalresponse,” Pharmacogenomics. 2000 February; 1(1):15-26).

SNPs are identified by a variety of art-accepted methods (P. Bean, “Thepromising voyage of SNP target discovery,” Am. Clin. Lab. 2001October-November; 20(9):18-20; K. M. Weiss, “In search of humanvariation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enablinglarge-scale pharmacogenetic studies by high-throughput mutationdetection and genotyping technologies,” Clin. Chem. 2001 February;47(2):164-172). For example, SNPs are identified by sequencing DNAfragments that show polymorphism by gel-based methods such asrestriction fragment length polymorphism (RFLP) and denaturing gradientgel electrophoresis (DGGE). They can also be discovered by directsequencing of DNA samples pooled from different individuals or bycomparing sequences from different DNA samples. With the rapidaccumulation of sequence data in public and private databases, one candiscover SNPs by comparing sequences using computer programs (Z. Gu, L.Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting incyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified andgenotype or haplotype of an individual can be determined by a variety ofmethods including direct sequencing and high throughput microarrays (P.Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu.Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K.Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft,“High-throughput SNP genotyping with the Masscode system,” Mol. Diagn.2000 December; 5(4):329-340).

Using the methods described above, fourteen SNPs were identified in thetranscript from clone GTH9, designated as STEAP-1 v.2, at positions 602(C/G), 386 (C/T), 1087 (T/G), 1447 (T/C), 1621 (A/T, 1625 (G/T, 1716(C/A), 2358 (C/T), 2646 (T/G), 2859 (T/G), 2908 (A/T), 3006 (G/C), 3107(C/T), and 3180 (A/T). The transcripts or proteins with alternativealleles were designated as variants STEAP-1 v.4, v.5, v.6, v.7, v.8,v.9, v.10, v.11, v.12, v.13, v.14, v.15, v.16 and v.17, respectively.FIG. 10 shows the schematic alignment of the SNP variants. FIG. 12 showsthe schematic alignment of protein variants, corresponding to nucleotidevariants. These alleles of the SNPs, though shown separately here, canoccur in different combinations (haplotypes) and in any one of thetranscript variants (such as STEAP-1 v.1 and v.3) that contains thesequence context of the SNPs. E.g., the first two SNP were also onSTEAP-1 v.3 at the same positions, but at 572 and 356, respectively, onSTEAP-1 v.1.

Example 7 Production of Recombinant STEAP-1 in Prokaryotic Systems

To express recombinant STEAP-1 and STEAP-1 variants in prokaryoticcells, the full or partial length STEAP-1 and STEAP-1 variant cDNAsequences are cloned into any one of a variety of expression vectorsknown in the art. The full length cDNA, or any 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morecontiguous amino acids from STEAP-1, variants, or analogs thereof areused.

A. In Vitro Transcription and Translation Constructs:

pCRII:

To generate STEAP-1 sense and anti-sense RNA probes for RNA in situinvestigations, pCRII constructs (Invitrogen, Carlsbad Calif.) aregenerated encoding either all or fragments of the STEAP-1 cDNA. ThepCRII vector has Sp6 and T7 promoters flanking the insert to drive thetranscription of STEAP-1 RNA for use as probes in RNA in situhybridization experiments. These probes are used to analyze the cell andtissue expression of STEAP-1 at the RNA level. Transcribed STEAP-1 RNArepresenting the cDNA amino acid coding region of the STEAP-1 gene isused in in vitro translation systems such as the TnT™ CoupledReticulolysate System (Promega, Corp., Madison, Wis.) to synthesizeSTEAP-1 protein.

B. Bacterial Constructs:

pGEX Constructs:

To generate recombinant STEAP-1 proteins in bacteria that are fused tothe Glutathione S-transferase (GST) protein, all or parts of the STEAP-1cDNA or variants are cloned into the GST-fusion vector of the pGEXfamily (Amersham Pharmacia Biotech, Piscataway, N.J.). These constructsallow controlled expression of recombinant STEAP-1 protein sequenceswith GST fused at the amino-terminus and a six histidine epitope (6×His)at the carboxyl-terminus. The GST and 6×His tags permit purification ofthe recombinant fusion protein from induced bacteria with theappropriate affinity matrix and allow recognition of the fusion proteinwith anti-GST and anti-His antibodies. The 6×His tag is generated byadding 6 histidine codons to the cloning primer at the 3′ end, e.g., ofthe open reading frame (ORF). A proteolytic cleavage site, such as thePreScission™ recognition site in pGEX-6P-1, may be employed such that itpermits cleavage of the GST tag from STEAP-1-related protein. Theampicillin resistance gene and pBR322 origin permits selection andmaintenance of the pGEX plasmids in E. coli.

pMAL Constructs:

To generate, in bacteria, recombinant STEAP-1 proteins that are fused tomaltose-binding protein (MBP), all or parts of the STEAP-1 cDNA proteincoding sequence are fused to the MBP gene by cloning into the pMAL-c2×and pMAL-p2× vectors (New England Biolabs, Beverly, Mass.). Theseconstructs allow controlled expression of recombinant STEAP-1 proteinsequences with MBP fused at the amino-terminus and a 6×His epitope tagat the carboxyl-terminus. The MBP and 6×His tags permit purification ofthe recombinant protein from induced bacteria with the appropriateaffinity matrix and allow recognition of the fusion protein withanti-MBP and anti-His antibodies. The 6×His epitope tag is generated byadding 6 histidine codons to the 3′ cloning primer. A Factor Xarecognition site permits cleavage of the pMAL tag from STEAP-1. ThepMAL-c2× and pMAL-p2× vectors are optimized to express the recombinantprotein in the cytoplasm or periplasm respectively. Periplasm expressionenhances folding of proteins with disulfide bonds.

pET Constructs:

To express STEAP-1 in bacterial cells, all or parts of the STEAP-1 cDNAprotein coding sequence are cloned into the pET family of vectors(Novagen, Madison, Wis.). These vectors allow tightly controlledexpression of recombinant STEAP-1 protein in bacteria with and withoutfusion to proteins that enhance solubility, such as NusA and thioredoxin(Trx), and epitope tags, such as 6×His and S-Tag™ that aid purificationand detection of the recombinant protein. For example, constructs aremade utilizing pET NusA fusion system 43.1 such that regions of theSTEAP-1 protein are expressed as amino-terminal fusions to NusA.

C. Yeast Constructs:

pESC Constructs:

To express STEAP-1 in the yeast species Saccharomyces cerevisiae forgeneration of recombinant protein and functional studies, all or partsof the STEAP-1 cDNA protein coding sequence are cloned into the pESCfamily of vectors each of which contain 1 of 4 selectable markers, HIS3,TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.). These vectors allowcontrolled expression from the same plasmid of up to 2 different genesor cloned sequences containing either Flag™ or Myc epitope tags in thesame yeast cell. This system is useful to confirm protein-proteininteractions of STEAP-1. In addition, expression in yeast yields similarpost-translational modifications, such as glycosylations andphosphorylations, that are found when expressed in eukaryotic cells.

pESP Constructs:

To express STEAP-1 in the yeast species Saccharomyces pombe, all orparts of the STEAP-1 cDNA protein coding sequence are cloned into thepESP family of vectors. These vectors allow controlled high level ofexpression of a STEAP-1 protein sequence that is fused at either theamino terminus or at the carboxyl terminus to GST which aidspurification of the recombinant protein. A Flag™ epitope tag allowsdetection of the recombinant protein with anti-Flag™ antibody.

Example 8 Production of Recombinant STEAP-1 in Higher Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant STEAP-1 in eukaryotic cells, the full or partiallength STEAP-1 cDNA sequences can be cloned into any one of a variety ofexpression vectors known in the art. One or more of the followingregions of STEAP-1 are expressed in these constructs, amino acids 1 to339 of STEAP-1 v.1, v.4, amino acids 1 to 258 of v.2, amino acids 1 to282 of v.3, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous aminoacids from STEAP-1, variants, or analogs thereof. In certain embodimentsa region of a specific variant of STEAP-1 is expressed that encodes anamino acid at a specific position which differs from the amino acid ofany other variant found at that position. In other embodiments, a regionof a variant of STEAP-1 is expressed that lies partly or entirely withina sequence that is unique to that variant.

The constructs can be transfected into any one of a wide variety ofmammalian cells such as 293T cells. Transfected 293T cell lysates can beprobed with the anti-STEAP-1 polyclonal serum, described herein.

pcDNA4/HisMax Constructs:

To express STEAP-1 in mammalian cells, a STEAP-1 ORF, or portionsthereof, of STEAP-1 are cloned into pcDNA4/HisMax Version A (Invitrogen,Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus(CMV) promoter and the SP16 translational enhancer. The recombinantprotein has Xpress™ and six histidine (6×His) epitopes fused to theamino-terminus. The pcDNA4/HisMax vector also contains the bovine growthhormone (BGH) polyadenylation signal and transcription terminationsequence to enhance mRNA stability along with the SV40 origin forepisomal replication and simple vector rescue in cell lines expressingthe large T antigen. The Zeocin resistance gene allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli.

pcDNA3.1/MycHis Constructs:

To express STEAP-1 in mammalian cells, a STEAP-1 ORF, or portionsthereof, of STEAP-1 with a consensus Kozak translation initiation sitewas cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad,Calif.). Protein expression is driven from the cytomegalovirus (CMV)promoter. The recombinant proteins have the myc epitope and 6×Hisepitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector alsocontains the bovine growth hormone (BGH) polyadenylation signal andtranscription termination sequence to enhance mRNA stability, along withthe SV40 origin for episomal replication and simple vector rescue incell lines expressing the large T antigen. The Neomycin resistance genewas used, as it allows for selection of mammalian cells expressing theprotein and the ampicillin resistance gene and ColE1 origin permitsselection and maintenance of the plasmid in E. coli.

pcDNA3.1/CT-GFP-TOPO Construct:

To express STEAP-1 in mammalian cells and to allow detection of therecombinant proteins using fluorescence, a STEAP-1 ORF, or portionsthereof, with a consensus Kozak translation initiation site are clonedinto pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is drivenfrom the cytomegalovirus (CMV) promoter. The recombinant proteins havethe Green Fluorescent Protein (GFP) fused to the carboxyl-terminusfacilitating non-invasive, in vivo detection and cell biology studies.The pcDNA3.1 CT-GFP-TOPO vector also contains the bovine growth hormone(BGH) polyadenylation signal and transcription termination sequence toenhance mRNA stability along with the SV40 origin for episomalreplication and simple vector rescue in cell lines expressing the largeT antigen. The Neomycin resistance gene allows for selection ofmammalian cells that express the protein, and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli. Additional constructs with an amino-terminal GFP fusion aremade in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a STEAP-1protein.

PAPtaq:

A STEAP-1 ORF, or portions thereof, is cloned into pAPtag-5 (GenHunterCorp. Nashville, Tenn.). This construct generates an alkalinephosphatase fusion at the carboxyl-terminus of a STEAP-1 protein whilefusing the IgG_(K) signal sequence to the amino-terminus. Constructs arealso generated in which alkaline phosphatase with an amino-terminalIgG_(K) signal sequence is fused to the amino-terminus of a STEAP-1protein. The resulting recombinant STEAP-1 proteins are optimized forsecretion into the media of transfected mammalian cells and can be usedto identify proteins such as ligands or receptors that interact withSTEAP-1 proteins. Protein expression is driven from the CMV promoter andthe recombinant proteins also contain myc and 6×His epitopes fused atthe carboxyl-terminus that facilitates detection and purification. TheZeocin resistance gene present in the vector allows for selection ofmammalian cells expressing the recombinant protein and the ampicillinresistance gene permits selection of the plasmid in E. coli.

ptag5:

A STEAP-1 ORF, or portions thereof, was cloned into pTag-5. This vectoris similar to pAPtag but without the alkaline phosphatase fusion. Thisconstruct generated STEAP-1 protein with an amino-terminal IgG_(K)signal sequence and myc and 6×His epitope tags at the carboxyl-terminusthat facilitate detection and affinity purification. The resultingrecombinant STEAP-1 protein was optimized for secretion into the mediaof transfected mammalian cells, and is used as immunogen or ligand toidentify proteins such as ligands or receptors that interact with theSTEAP-1 proteins. Protein expression was driven from the CMV promoter.The Zeocin resistance gene present in the vector allowed for selectionof mammalian cells expressing the protein, and the ampicillin resistancegene permits selection of the plasmid in E. coli.

PsecFc:

A STEAP-1 ORF, or portions thereof, was also cloned into psecFc. ThepsecFc vector was assembled by cloning the human immunoglobulin G1 (IgG)Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California).This construct generated an IgG1 Fc fusion at the carboxyl-terminus ofthe STEAP-1 proteins, while fusing the IgGK signal sequence toN-terminus. STEAP-1 fusions utilizing the murine IgG1 Fc region are alsoused. The resulting recombinant STEAP-1 proteins were optimized forsecretion into the media of transfected mammalian cells, and can wereused as immunogens or to identify proteins such as ligands or receptorsthat interact with STEAP-1 protein. Protein expression is driven fromthe CMV promoter. The hygromycin resistance gene present in the vectorallowed for selection of mammalian cells that express the recombinantprotein, and the ampicillin resistance gene permits selection of theplasmid in E. coli.

pSRα Constructs:

To generate mammalian cell lines that express STEAP-1 constitutively,STEAP-1 ORF, or portions thereof, of STEAP-1 were cloned into pSRaconstructs. Amphotropic and ecotropic retroviruses were generated bytransfection of pSRa constructs into the 293T-10A1 packaging line orco-transfection of pSRa and a helper plasmid (containing deletedpackaging sequences) into the 293 cells, respectively. The retroviruswas used to infect a variety of mammalian cell lines, resulting in theintegration of the cloned gene, STEAP-1, into the host cell-lines.Protein expression was driven from a long terminal repeat (LTR). TheNeomycin resistance gene present in the vector allowed for selection ofmammalian cells that express the protein, and the ampicillin resistancegene and ColE1 origin permit selection and maintenance of the plasmid inE. coli. The retroviral vectors were thereafter be used for infectionand generation of various cell lines using, for example, PC3, NIH 3T3,TsuPr1, 293 or rat-1 cells.

Additional pSRα constructs are made that fuse an epitope tag such as theFLAG™ tag to the carboxyl-terminus of STEAP-1 sequences to allowdetection using anti-Flag antibodies. For example, the FLAG™ sequence 5′gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: 80) is added to cloningprimer at the 3′ end of the ORF. Additional pSRα constructs were made toproduce both amino-terminal and carboxyl-terminal GFP and myc/6×Hisfusion proteins of the full-length STEAP-1 proteins.

Additional Viral Vectors:

Additional constructs are made for viral-mediated delivery andexpression of STEAP-1. High virus titer leading to high level expressionof STEAP-1 is achieved in viral delivery systems such as adenoviralvectors and herpes amplicon vectors. A STEAP-1 coding sequences orfragments thereof are amplified by PCR and subcloned into the AdEasyshuffle vector (Stratagene). Recombination and virus packaging areperformed according to the manufacturers instructions to generateadenoviral vectors. Alternatively, STEAP-1 coding sequences or fragmentsthereof are cloned into the HSV-1 vector (Imgenex) to generate herpesviral vectors. The viral vectors are thereafter used for infection ofvarious cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

Regulated Expression Systems:

To control expression of STEAP-1 in mammalian cells, coding sequences ofSTEAP-1, or portions thereof, are cloned into regulated mammalianexpression systems such as the T-Rex System (Invitrogen), the GeneSwitchSystem (Invitrogen) and the tightly-regulated Ecdysone System(Sratagene). These systems allow the study of the temporal andconcentration dependent effects of recombinant STEAP-1. These vectorsare thereafter used to control expression of STEAP-1 in various celllines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems

To generate recombinant STEAP-1 proteins in a baculovirus expressionsystem, STEAP-1 ORF, or portions thereof, are cloned into thebaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus. Specifically, pBlueBac-STEAP-1 isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.Recombinant STEAP-1 protein is then generated by infection of HighFiveinsect cells (Invitrogen) with purified baculovirus. Recombinant STEAP-1protein can be detected using anti-STEAP-1 or anti-His-tag antibody.STEAP-1 protein can be purified and used in various cell-based assays oras immunogen to generate polyclonal and monoclonal antibodies specificfor STEAP-1.

Example 9 Antictenicity Profiles and Secondary Structure

FIGS. 5( a)-9(a) and 5(b)-9(b) depict graphically five amino acidprofiles of the STEAP-1 variants 1 and 3 respectively, each assessmentavailable by accessing the ProtScale website located on the World WideWeb at (URL.expasy.ch/cgi-bin/protscale.p1) on the ExPasy molecularbiology server.

These profiles: FIGS. 5( a) and (b), Hydrophilicily, (Hopp T. P., WoodsK. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIGS. 6( a)and (b), Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol.157:105-132); FIGS. 7( a) and (b), Percentage Accessible Residues (JaninJ., 1979 Nature 277:491-492); FIGS. 8( a) and (b), Average Flexibility,(Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res.32:242-255); FIGS. 9( a) and (b), Beta-turn (Deleage, G., Roux B. 1987Protein Engineering 1:289-294); and optionally others available in theart, such as on the ProtScale website, were used to identify antigenicregions of the STEAP-1 protein. Each of the above amino acid profiles ofSTEAP-1 were generated using the following ProtScale parameters foranalysis: 1) A window size of 9; 2) 100% weight of the window edgescompared to the window center; and, 3) amino acid profile valuesnormalized to lie between 0 and 1.

Hydrophilicity (FIGS. 5( a) and (b)), Hydropathicity (FIGS. 6( a) and(b)) and Percentage Accessible Residues (FIGS. 7( a) and (b)) profileswere used to determine stretches of hydrophilic amino acids (i.e.,values greater than 0.5 on the Hydrophilicity and Percentage AccessibleResidues profile, and values less than 0.5 on the Hydropathicityprofile). Such regions are likely to be exposed to the aqueousenvironment, be present on the surface of the protein, and thusavailable for immune recognition, such as by antibodies.

Average Flexibility (FIGS. 8( a) and (b)) and Beta-turn (FIGS. 9( a) and(b)) profiles determine stretches of amino acids (i.e., values greaterthan 0.5 on the Beta-turn profile and the Average Flexibility profile)that are not constrained in secondary structures such as beta sheets andalpha helices. Such regions are also more likely to be exposed on theprotein and thus accessible to immune recognition, such as byantibodies.

Antigenic sequences of the STEAP-1 protein and of the variant proteinsindicated, e.g., by the profiles set forth in FIGS. 5( a) and (b), FIGS.6( a) and (b), FIGS. 7( a) and (b), FIGS. 8( a) and (b), and/or FIGS. 9(a) and (b) are used to prepare immunogens, either peptides or nucleicacids that encode them, to generate therapeutic and diagnosticanti-STEAP-1 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,50 or more than 50 contiguous amino acids, or the corresponding nucleicacids that encode them, from the STEAP-1 protein variants listed inFIGS. 2 and 3. In particular, peptide immunogens of the invention cancomprise, a peptide region of at least 5 amino acids of FIGS. 2 and 3 inany whole number increment that includes an amino acid position having avalue greater than 0.5 in the Hydrophilicity profile of FIGS. 5( a) and(b); a peptide region of at least 5 amino acids of FIGS. 2 and 3 in anywhole number increment that includes an amino acid position having avalue less than 0.5 in the Hydropathicity profile of FIGS. 6( a) and(b); a peptide region of at least 5 amino acids of FIGS. 2 and 3 in anywhole number increment that includes an amino acid position having avalue greater than 0.5 in the Percent Accessible Residues profile ofFIGS. 7( a) and (b); a peptide region of at least 5 amino acids of FIGS.2 and 3 in any whole number increment that includes an amino acidposition having a value greater than 0.5 in the Average Flexibilityprofile on FIGS. 8( a) and (b); and, a peptide region of at least 5amino acids of FIGS. 2 and 3 in any whole number increment that includesan amino acid position having a value greater than 0.5 in the Beta-turnprofile of FIGS. 9( a) and (b). Peptide immunogens of the invention canalso comprise nucleic acids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can beembodied in human unit dose form, or comprised by a composition thatincludes a pharmaceutical excipient compatible with human physiology.

The secondary structures of STEAP-1 variant 1 and variant 3, namely thepredicted presence and location of alpha helices, extended strands, andrandom coils, are predicted from the respective primary amino acidsequences using the HNN—Hierarchical Neural Network method (Guermeur,1997, http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html),accessed from the ExPasy molecular biology server located on the WorldWide Web at (.expasy.ch/tools/). The analysis indicates that STEAP-1variant 1 is composed of 64.60% alpha helix, 4.72% extended strand, and30.68% random coil (FIG. 13 a). STEAP-1 variant 2 is composed of 62.79%alpha helix, 3.10% extended strand, and 34.11% random coil (FIG. 13 b).STEAP-1 variant 3 is composed of 58.87% alpha helix, 5.32% extendedstrand, and 35.82% random coil (FIG. 13 c).

Analysis for the potential presence of transmembrane domains in STEAP-1variants were carried out using a variety of transmembrane predictionalgorithms accessed from the ExPasy molecular biology server located onthe World Wide Web at (.expasy.ch/tools/). Shown graphically are theresults of analysis of variant 1 depicting the presence and location of6 transmembrane domains using the TMpred program (FIG. 13 d) and TMHMMprogram (FIG. 13 e). Also shown are the results of analysis of variant 2depicting the presence and location of 4 transmembrane domains usingTMpred (FIGS. 13 f) and 3 transmembrane domains using TMHMM (FIG. 13 g).Analysis of variant 3 predicts the presence of 4 transmembrane domainsusing the TMpred (FIG. 13 h) and 3 transmembrane domains with TMHMM(FIG. 13 i). The results of each program, namely the amino acidsencoding the transmembrane domains are summarized in Table XX.

Example 10 Generation of STEAP-1 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Inaddition to immunizing with a full length STEAP-1 protein variant,computer algorithms are employed in design of immunogens that, based onamino acid sequence analysis contain characteristics of being antigenicand available for recognition by the immune system of the immunized host(see the Example entitled “Antigenicity Profiles and SecondaryStructure”). Such regions would be predicted to be hydrophilic,flexible, in beta-turn conformations, and be exposed on the surface ofthe protein (see, e.g., FIGS. 5( a) and (b), FIGS. 6( a) and (b), FIGS.7( a) and (b), FIGS. 8( a) and (b), and/or FIGS. 9( a) and (b) for aminoacid profiles that indicate such regions of STEAP-1 protein variants 1and 3).

For example, recombinant bacterial fusion proteins or peptidescontaining hydrophilic, flexible, beta-turn regions of STEAP-1 proteinvariants are used as antigens to generate polyclonal antibodies in NewZealand White rabbits or monoclonal antibodies as described in exampleentitled (“Generation of STEAP-1 Monoclonal Antibodies (MAbs). Forexample, such regions include, but are not limited to, amino acids 1-40,amino acids 143-165, amino acids 180-220, of STEAP-1 variants 1, 2, and3, amino acids 312-339 of STEAP-1 variant 1, and amino acids 250-282 ofSTEAP-1 variant 3. It is useful to conjugate the immunizing agent to aprotein known to be immunogenic in the mammal being immunized. Examplesof such immunogenic proteins include, but are not limited to, keyholelimpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, andsoybean trypsin inhibitor. In one embodiment, a peptide encoding aminoacids 250-282 of STEAP-1 variant 3 is conjugated to KLH. This peptide isthen used as immunogen. Alternatively the immunizing agent may includeall or portions of the STEAP-1 variant proteins, analogs or fusionproteins thereof. For example, the STEAP-1 variant 1 amino acid sequencecan be fused using recombinant DNA techniques to any one of a variety offusion protein partners that are well known in the art, such asglutathione-S-transferase (GST) and HIS tagged fusion proteins. Inanother embodiment, amino acids 250-282 of STEAP-1 variant 1 is fused toGST using recombinant techniques and the pGEX expression vector,expressed, purified and used to immunize a rabbit. Such fusion proteinsare purified from induced bacteria using the appropriate affinitymatrix.

Other recombinant bacterial fusion proteins that may be employed includemaltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulinconstant region (see the section entitled “Production of STEAP-1 inProkaryotic Systems” and Current Protocols In Molecular Biology, Volume2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P.S.,Brady, W., Umes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991)J. Exp. Med. 174, 561-566).

In addition to bacterial derived fusion proteins, mammalian expressedprotein antigens are also used. These antigens are expressed frommammalian expression vectors such as the Tag5 and Fc-fusion vectors (seethe section entitled “Production of Recombinant STEAP-1 in EukaryoticSystems”), and retain post-translational modifications such asglycosylations found in native protein. In one embodiment, amino acids185-218 of STEAP-1 variant 1 were cloned into the Tag5 mammaliansecretion vector, and expressed in 293T cells. The recombinant proteinwas purified by metal chelate chromatography from tissue culturesupernatants of 293T cells stably expressing the recombinant vector. Thepurified Tag5 STEAP-1 variant 1 protein is then used as immunogen.

During the immunization protocol, it is useful to mix or emulsify theantigen in adjuvants that enhance the immune response of the hostanimal. Examples of adjuvants include, but are not limited to, completeFreund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneouslywith up to 200 μg, typically 100-200 μg, of fusion protein or peptideconjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits arethen injected subcutaneously every two weeks with up to 200 μg,typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant(IFA). Test bleeds are taken approximately 7-10 days following eachimmunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbitserum derived from immunization with the GST-fusion of STEAP-1 variant 1protein, the full-length STEAP-1 variant 1 cDNA is cloned into pCDNA 3.1myc-his expression vector (Invitrogen, see the Example entitled“Production of Recombinant STEAP-1 in Eukaryotic Systems”). Aftertransfection of the constructs into 293T cells, cell lysates are probedwith the anti-STEAP-1 serum and with anti-His antibody (Santa CruzBiotechnologies, Santa Cruz, Calif.) to determine specific reactivity todenatured STEAP-1 protein using the Western blot technique. In addition,the immune serum is tested by fluorescence microscopy, flow cytometryand immunoprecipitation against 293T and other recombinantSTEAP-1-expressing cells to determine specific recognition of nativeprotein. Western blot, immunoprecipitation, fluorescent microscopy, andflow cytometric techniques using cells that endogenously express STEAP-1are also carried out to test reactivity and specificity.

Anti-serum from rabbits immunized with STEAP-1 variant fusion proteins,such as GST and MBP fusion proteins, are purified by depletion ofantibodies reactive to the fusion partner sequence by passage over anaffinity column containing the fusion partner either alone or in thecontext of an irrelevant fusion protein. For example, antiserum derivedfrom a GST-STEAP-1 variant 1 fusion protein is first purified by passageover a column of GST protein covalently coupled to AffiGel matrix(BioRad, Hercules, Calif.). The antiserum is then affinity purified bypassage over a column composed of a MBP-STEAP-1 fusion proteincovalently coupled to Affigel matrix. The serum is then further purifiedby protein G affinity chromatography to isolate the IgG fraction. Serafrom other His-tagged antigens and peptide immunized rabbits as well asfusion partner depleted sera are affinity purified by passage over acolumn matrix composed of the original protein immunogen or freepeptide.

Example 11 Generation of STEAP-1 Monoclonal Antibodies (MAbs)

In one embodiment, therapeutic MAbs to STEAP-1 variants comprise thosethat react with epitopes specific for each variant protein or specificto sequences in common between the variants that would bind,internalize, disrupt or modulate the biological function of the STEAP-1variants, for example those that would disrupt the interaction withligands and binding partners. Immunogens for generation of such MAbsinclude those designed to encode or contain the extracellular domain orthe entire STEAP-1 protein variant sequence, regions predicted tocontain functional motifs, and regions of the STEAP-1 protein variantspredicted to be antigenic from computer analysis of the amino acidsequence (see, e.g., FIG. 5( a)-(b), FIG. 6( a)-(b), FIG. 7( a)-(b),FIG. 8( a)-(b), or FIG. 9( a)-(b), and the Example entitled“Antigenicity Profiles and Secondary Structure”). Immunogens includepeptides, recombinant bacterial proteins, and mammalian expressed Tag 5proteins and human and murine IgG FC fusion proteins. In addition, pTAG5protein, DNA vectors encoding the pTAG5 cells engineered to express highlevels of a respective STEAP-1 variant, such as 293T-STEAP-1 variant 1or 3T3, RAT, or 300.19-STEAP-1 variant 1 murine Pre-B cells, are used toimmunize mice.

To generate MAbs to STEAP-1 variants, mice are first immunizedintraperitoneally (IP) or in the foot pad with, typically, 10-50 μg ofprotein immunogen or 10⁷ STEAP-1-expressing cells mixed in completeFreund's adjuvant. Examples of other adjuvants used are Titermax (Sigma)and Immuneasy (Qiagen). Mice are then subsequently immunized IP every2-4 weeks with, typically, 10-50 ptg of protein immunogen or 10⁷ cellsmixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvantis used in immunizations. In addition to the above protein andcell-based immunization strategies, a DNA-based immunization protocol isemployed in which a mammalian expression vector encoding a STEAP-1variant sequence is used to immunize mice by direct injection of theplasmid DNA. For example, amino acids 185-218 of STEAP-1 of variant 1was cloned into the Tag5 mammalian secretion vector and the recombinantvector was used as immunogen. In another example, the same amino acidswere cloned into an Fc-fusion secretion vector in which the STEAP-1variant 1 sequence is fused at the amino-terminus to an IgK leadersequence and at the carboxyl-terminus to the coding sequence of thehuman or murine IgG Fc region. This recombinant vector was then used asimmunogen. The plasmid immunization protocols were used in combinationwith purified proteins expressed from the same vector and with cellsexpressing the respective STEAP-1 variant. In another example, amonoclonal antibody to STEAP-1 variant 3 is generated by using a peptideencoding amino acids 250-282. The peptide is conjugated to KLH and usedas immunogen. ELISA on free peptide is used to identify immunoreactiveclones. Reactivity and specificity of the monoclonal antibodies to fulllength STEAP-1 variant 1 protein is monitored by Western blotting,immunoprecipitation, and flow cytometry using both recombinant andendogenous-expressing STEAP-1 variant 1 cells.

During the immunization protocol, test bleeds are taken 7-10 daysfollowing an injection to monitor titer and specificity of the immuneresponse. Once appropriate reactivity and specificity is obtained asdetermined by ELISA, Western blotting, immunoprecipitation, fluorescencemicroscopy, and flow cytometric analyses, fusion and hybridomageneration is then carried out with established procedures well known inthe art (see, e.g., Harlow and Lane, 1988).

The binding affinity of STEAP-1 variant 1 specific monoclonal antibodieswas determined using standard technologies. Affinity measurementsquantify the strength of antibody to epitope binding and are used tohelp define which STEAP-1 variant monoclonal antibodies preferred fordiagnostic or therapeutic use, as appreciated by one of skill in theart. The BIAcore system (Uppsala, Sweden) is a preferred method fordetermining binding affinity. The BIAcore system uses surface plasmonresonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton andMyszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecularinteractions in real time. BIAcore analysis conveniently generatesassociation rate constants, dissociation rate constants, equilibriumdissociation constants, and affinity constants.

To generate monoclonal antibodies specific for other STEAP-1 variants,immunogens are designed to encode amino acid sequences unique to thevariants. In one embodiment, a peptide encoding amino acids unique toSTEAP-1 variants are synthesized, coupled to KLH, and used as immunogen.In another embodiment, peptides or bacterial fusion proteins are madethat encompass the unique sequence generated by alternative splicing inthe variants. Hybridomas are then selected that recognize the respectivevariant specific antigen and also recognize the full length variantprotein expressed in cells. Such selection utilizes immunoassaysdescribed above such as Western blotting, immunoprecipitation, and flowcytometry.

In one embodiment, the invention provides for monoclonal antibodiesdesignated X92.1.30.1.1(1) (a.k.a. M2/92.30) and X120.545.1.1 (a.k.a.M2.120.545). M2/92.30 and M2/120.545 were identified and are shown toreact and bind with cell surface STEAP-1 (See, FIGS. 15 and 18). FIG. 16shows that the anti-STEAP-1 MAb M2/92.30 binds endogenous cell surfaceSTEAP-1 expressed in bladder and prostate cancer xenograft cells.Additionally, M2/92.30 reacts and binds with murine STEAP-1 as shown inFIG. 17.

The antibodies designated X92.1.30.1.1(1) (a.k.a. M2/92.30) andX120.545.1.1 (a.k.a. M2.120.545) were sent (via Federal Express) to theAmerican Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Va.20108 on 6 Feb. 2004 and assigned Accession numbers PTA-5802 andPTA-5803 respectively.

To clone the M2/X92.30 and M2/X120.545 antibodies the followingprotocols were used. Hybridoma cells were lysed with Trizol reagent(Life Technologies, Gibco BRL). Total RNA was purified and quantified.First strand cDNAs was generated from total RNA with oligo (dT)12-18priming using the Gibco-BRL Superscript Preamplification system. PCRproducts were cloned into the pCRScript vector (Stratagene, La Jolla).Several clones were sequenced and the variable heavy (“VH”) and variablelight (“VL”) chain regions determined. The nucleic acid and amino acidsequences of M2/X92.30 and M2/X120.545 variable heavy and light chainregions are listed in FIG. 19( a)-19(d) and FIG. 20( a)-(e).

Example 12 Characterization of STEAP-1 Antibodies

A. Cell Surface Binding

Reactivity of STEAP-1 antibodies with a STEAP-1-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,STEAP-1-related proteins, STEAP-1-expressing cells or extracts thereof.As shown in FIG. 15 FACS analysis of recombinant 3T3 and Rat-1 cellsstably expressing either STEAP-1 or a control stained with anti-STEAPMAb M2/92.30 (10 ug/ml) and cell surface bound MAb was detected with agoat anti-mouse IgG-PE conjugate secondary reagent. The stained cellswere then subjected to FACS analysis. As indicated by the fluorescentshift of the Rat1-STEAP1 and 3T3-STEAP1 cells compared to the respectivecontrol cells, M2/92.30 specifically binds to cell surface STEAP1.

In addition, when UGB1 bladder cancer cells and LAPC9 prostate cancercells were stained with 10 ug/ml of either MAb M2/92.30 or with acontrol anti-KLH MAb. Surface bound MAb 92.30 was detected withgoat-anti-mouse IgG-PE conjugated secondary Ab. Stained cells were thensubjected to FACS analysis. These results demonstrate that theanti-STEAP1 MAb M2/92.30 specifically binds endogenous cell surfaceSTEAP1 expressed in bladder and prostate cancer xenograft cells (FIG.21).

STEAP-1 M2/92.30 is also shown to bind to murine STEAP-1 protein (SeeFIG. 17). In this experiment 293T cells were transiently transfectedwith either pCDNA3.1 encoding the murine STEAP1 cDNA or with an emptyvector. 48 hours later, the cells were harvested and stained withanti-STEAP1 MAb M2/92.30 (10 ug/ml) and cell surface bound MAb 92.30 wasdetected with a goat anti-mouse IgG-PE conjugate secondary reagent.Cells were then subjected to FACS analysis. STEAP-1 M2/92.30 was shownto bind specifically to STEAP-1-expressed 293T cells.

STEAP-1 M2/120.545 is also shown to specifically bind to STEAP-1 (SeeFIG. 18). 3T3-neo (Panel A, filled histograms) and 3T3-STEAP1 cells(Panel A, no fill histograms) and Rat1-neo (Panel B, filled histograms)and Rat1-STEAP cells (Panel B, no fill histograms) were stained with MAbM2/120.545 (10 ug/ml) and surface bound MAb was detected with goatanti-mouse IgG-PE conjugated secondary Ab. Cells were then subjected toFACS analysis. As indicated by the fluorescence shift of the 3T3-STEAP1and Rat1-STEAP1 cells compared to their respective neo controls, MAbM2/120.545 specifically binds cell surface STEAP1. In Panel C, LNCaPcells were stained with either MAb M2/120.545 or a control anti-KLH MAband subjected to FACS analysis as above. In Panel D, Fluorescencemicroscopy of the M2/120.545 stained LNCaP cells showing bright cellsurface fluorescence.

Reactivity and specificity of M2/92.30 and M2/120.545 were alsodetermined by immunoprecipitation. FIG. 25 shows 3T3-STEAP1 and 3T3-neocells were lysed in RIPA buffer (25 mM Tris-CI pH7.4; 150 mM NaCl, 0.5mM EDTA, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, and proteaseinhibitor cocktail). The cell lysates were precleared with protein Gsepharose beads and then incubated with 5 ug of either MAb M2/92.30 orM2/120.545 for 2 hours at room temperature. Protein G beads were addedand the mixture was further incubated for 1 hour. The immune complexeswere washed and solubilized in SDS-PAGE sample buffer. The solubilizedsamples were then subjected to SDS-PAGE and Western blot analysis usinga rabbit anti-STEAP pAb. Whole cell lysates of 293T cells transfectedwith STEAP1 was also run as a positive control. An immunoreactive bandof ˜37 kD was seen only in samples derived from 3T3-STEAP1 cellsindicative of specific immunoprecipitation of STEAP1 by both M2/92.30and M2/120.545 MAbs.

B. STEAP-1 Antibody Internalization

Immunotherapy based on the delivery of toxins towards specific celltargets using monoclonal antibodies is considered a modality in thetherapy of malignancies. The general principle is the delivery of toxinsor antineoplastic drugs to cancer cells with molecules that bind toantigens or receptors that are either uniquely expressed oroverexpressed on the target cells relative to normal tissues.

Immunotoxins consist of cell selective ligands (usually monoclonalantibodies or cytokines) linked covalently to toxins. The interaction ofantibody or ligand with cell surface receptors triggers internalization.In defined intracellular vesicle compartments, the toxin moiety escapesto the cytosol, where it catalytically alters critical cell functionsleading to cell death. See, Frankel A E., Increased Sophistication ofImmunotoxins, Clinical cancer research 8: 942-944, (2002) and Allen™,Ligand-Targeted Therapeutics in Anti-cancer Therapy. Nature Reviews.2:750-760, (2002).

Saporin is a ribosome-inactivating protein (RIP) that catalyzes the invitro depurination of a specific adenine residue in large ribosomalRNAs. EndoY, et. al., Mechanism of Action of the Toxin Lectin Ricin onEukaryotic Cells; The Site and Characteristics of the Modification in28S RNA Caused by the Toxin, J. Biol. Chem. 262, 5908-5912, (1987). Itusually cannot enter cells unless complexed to an appropriate carriermolecule. Covalent conjugation of saporin to monoclonal antibodies thatrecognize tumor antigens produces immunotoxins that possess both cancercell selectivity and are internalized. See, Flavell, D J, SapoinImmunotoxins, Curr. Top. Microbiol. Immunol. 234: 51-61, (1998) andFlavell D J, et. al., Therapy of Human T-cell Acute LymphoblasticLeukemia with a Combination of Anti-CD7 and Anti-CD38-SaporinImmunotoxins is Significantly Better than Therapy with Each IndividualImmunotoxins. Br. J. Cancer 84: 571-578, (2001). These molecules haverecently entered phase I clinical trails for leukemia and multiplemyeloma. Foon K A. Monoclonal Antibody Therapies for Lymohomas. CancerJ. 6: 273-278, (2000).

The internalization of STEAP-1 M2/92.30 is shown in FIG. 22. In thisexperiment, PC3-STEAP1 cells were stained at 4 degrees C. withM2/120.545 MAb (10 ug/ml), washed, then incubated with goat anti-mouseIgG-PE conjugate secondary Ab. One-half of the cells were moved to 37degrees C. for 30 minutes and the other half remained at 4 degrees C.Cells from each treatment were then subjected to fluorescent microscopy.Cells that remained at 4 degrees C. showed bright staining on thecircumference of the cell surface. Cells that were moved to 37 degreesC. showed loss of the staining on the cell circumference and theappearance of punctate and aggregated fluorescence indicative of cappingand internalization.

STEAP-1 internalization by STEAP1 M2/120.545 MAb is shown in FIG. 23.PC3-STEAP1 cells were stained at 4 C with M2/120.545 MAb (10 ug/ml),washed, then incubated with goat anti-mouse IgG-PE conjugate secondaryAb. One-half of the cells were moved to 37 C for 30 minutes and theother half remained at 4 C. Cells from each treatment were thensubjected to fluorescent microscopy. Cells that remained at 4 C showedbright “ring-like” cell surface fluorescence. Cells that were moved to37 C showed loss of the “ring-like” cell surface fluorescence and theappearance of punctate and aggregated fluorescence indicative of cappingand internalization.

One approach for selecting appropriate antibody candidates forimmunotoxin delivery employs killing with a secondary antibodyconjugated with a drug or toxin molecule. The secondary conjugatedantibody piggybacks onto the primary antibody allowing the evaluation ofthe primary antibody to internalize and traffic to appropriateintracellular compartments. Once the conjugate is internalized, saporinbreaks away from the targeting agent and inactivates the ribosomes toeliminate target cells. Kohls M D and Lappi D A. MAb-ZAP: A Tool forEvaluating Antibody Efficacy for Use in an Immunotoxin. Bio Techniques.28(1): 162-165 (2000).

To select the appropriate antibody candidate using the above approach, asecondary immunotoxin, anti-mouse IgG—saporin conjugates (AdvancedTargeting Systems, San Diego, Calif.) was used to demonstrate thatmurine Steap-1 M2/120.545 enters target cells via expression of Steap-1on the cell surface of LNCaP cell. The following protocols were used.LNCap cells were plated at 5000 cells/90 μl/well in 96-well plate andincubated overnight. Second immunotoxin conjugates (anti-mouseIgG-saporine and anti-goat IgG-saporin) and anti-mouse IgG were made incell medium containing the final concentration at 100 ng/ml. 10 μl wereadded to each well. The primary antibody is added at the concentrationfrom 1-1000 ng/ml. The plates were incubated 72 hours and the viabilitywas determined by MTT assay. The results in FIG. 24 show that LNCaPcells were killed in the presence of anti-mouse IgG-saporin. No effectswere detected with either the secondary antibody alone (anti-mouse IgG)or nonspecific secondary antibody conjugates (anti-goat IgG saporin). Notoxicity was observed with the primary antibody (M2/120.545) alonetested up to 1 μg/ml.

Example 13 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules areperformed in accordance with disclosed protocols (e.g., PCT publicationsWO 94/20127 and WO 94/03205; Sidney et al., Current Protocols inImmunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995);Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHCmolecules (5 to 500 nM) are incubated with various unlabeled peptideinhibitors and 1-10 nM ¹²⁵1-radiolabeled probe peptides as described.Following incubation, MHC-peptide complexes are separated from freepeptide by gel filtration and the fraction of peptide bound isdetermined. Typically, in preliminary experiments, each MHC preparationis titered in the presence of fixed amounts of radiolabeled peptides todetermine the concentration of HLA molecules necessary to bind 10-20% ofthe total radioactivity. All subsequent inhibition and direct bindingassays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC₅₀≧HILA], the measuredIC₅₀ values are reasonable approximations of the true K_(D) values.Peptide inhibitors are typically tested at concentrations ranging from120 μg/ml to 1.2 ng/ml, and are tested in two to four completelyindependent experiments. To allow comparison of the data obtained indifferent experiments, a relative binding figure is calculated for eachpeptide by dividing the IC₅₀ of a positive control for inhibition by theIC₅₀ for each tested peptide (typically unlabeled versions of theradiolabeled probe peptide). For database purposes, and inter-experimentcomparisons, relative binding values are compiled. These values cansubsequently be converted back into IC₅₀ nM values by dividing the IC₅₀nM of the positive controls for inhibition by the relative binding ofthe peptide of interest. This method of data compilation is accurate andconsistent for comparing peptides that have been tested on differentdays, or with different lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotifand/or HLA motif-bearing peptides (see Table IV).

Example 14 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expressionplasmid. Minigene plasmids may, of course, contain variousconfigurations of B cell, CTL and/or HTL epitopes or epitope analogs asdescribed herein.

A minigene expression plasmid typically includes multiple CTL and HTLpeptide epitopes. In the present example, HLA-A2, -A3, -B7supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearingpeptide epitopes are used in conjunction with DR supermotif-bearingepitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearingpeptide epitopes derived STEAP-1, are selected such that multiplesupermotifs/motifs are represented to ensure broad population coverage.Similarly, HLA class II epitopes are selected from STEAP-1 to providebroad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearingepitopes and HLA DR-3 motif-bearing epitopes are selected for inclusionin the minigene construct. The selected CTL and HTL epitopes are thenincorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTLepitopes to the endoplasmic reticulum. For example, the Ii protein maybe fused to one or more HTL epitopes as described in the art, whereinthe CLIP sequence of the li protein is removed and replaced with an HLAclass II epitope sequence so that HLA class II epitope is directed tothe endoplasmic reticulum, where the epitope binds to an HLA class IImolecules.

This example illustrates the methods to be used for construction of aminigene-bearing expression plasmid. Other expression vectors that maybe used for minigene compositions are available and known to those ofskill in the art.

The minigene DNA plasmid of this example contains a consensus Kozaksequence and a consensus murine kappa 1 g-tight chain signal sequencefollowed by CTL and/or HTL epitopes selected in accordance withprinciples disclosed herein. The sequence encodes an open reading framefused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70nucleotides in length with 15 nucleotide overlaps, are synthesized andHPLC-purified. The oligonucleotides encode the selected peptide epitopesas well as appropriate linker nucleotides, Kozak sequence, and signalsequence. The final multiepitope minigene is assembled by extending theoverlapping oligonucleotides in three sets of reactions using PCR. APerkin/Elmer 9600 PCR machine is used and a total of 30 cycles areperformed using the following conditions: 95° C. for 15 sec, annealingtemperature (5° below the lowest calculated Tm of each primer pair) for30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCRreaction, 5 μg of each of two oligonucleotides are annealed andextended: In an example using eight oligonucleotides, i.e., four pairsof primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM(NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer products are gel-purified, and two reactionscontaining the product of 1+2 and 3+4, and the product of 5+6 and 7+8are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct. The full-length product is gel-purified and cloned intopCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 15 The Plasmid Construct and the Degree to which it InducesImmunogenicity

The degree to which a plasmid construct, for example a plasmidconstructed in accordance with the previous Example, is able to induceimmunogenicity is confirmed in vitro by determining epitope presentationby APC following transduction or transfection of the APC with anepitope-expressing nucleic acid construct. Such a study determines“antigenicity” and allows the use of human APC. The assay determines theability of the epitope to be presented by the APC in a context that isrecognized by a T cell by quantifying the density of epitope-HLA class Icomplexes on the cell surface. Quantitation can be performed by directlymeasuring the amount of peptide eluted from the APC (see, e.g., Slits etal., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684,1989); or the number of peptide-HLA class I complexes can be estimatedby measuring the amount of lysis or lymphokine release induced bydiseased or transfected target cells, and then determining theconcentration of peptide necessary to obtain equivalent levels of lysisor lymphokine release (see, e.g., Kageyama et al., J. Immunol.154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injectionsinto mice and subsequent in vitro assessment of CTL and HTL activity,which are analyzed using cytotoxicity and proliferation assays,respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761,1994.

For example, to confirm the capacity of a DNA minigene constructcontaining at least one HLA-A2 supermotif peptide to induce CTLs invivo, HLA-A2.1/K^(b) transgenic mice, for example, are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide as they would beencoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of therespective compositions (peptide epitopes encoded in the minigene or thepolyepitopic peptide), then assayed for peptide-specific cytotoxicactivity in a ⁵¹Cr release assay. The results indicate the magnitude ofthe CTL response directed against the A2-restricted epitope, thusindicating the in vivo immunogenicity of the minigene vaccine andpolyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responsesdirected toward the HLA-A2 supermotif peptide epitopes as does thepolyepitopic peptide vaccine. A similar analysis is also performed usingother HLA-A3 and HLA-B7 transgenic mouse models to assess CTL inductionby HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is alsofound that the minigene elicits appropriate immune responses directedtoward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene toinduce HTLs in vivo, DR transgenic mice, or for those epitopes thatcross react with the appropriate mouse MHC molecule, I-A^(b)-restrictedmice, for example, are immunized intramuscularly with 100 μg of plasmidDNA. As a means of comparing the level of HTLs induced by DNAimmunization, a group of control animals is also immunized with anactual peptide composition emulsified in complete Freund's adjuvant.CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunizedanimals and stimulated with each of the respective compositions(peptides encoded in the minigene). The HTL response is measured using a³H-thymidine incorporation proliferation assay, (see, e.g., Alexander etal. Immunity 1:751-761, 1994). The results indicate the magnitude of theHTL response, thus demonstrating the in vivo immunogenicity of theminigene.

DNA minigenes, constructed as described in the previous Example, canalso be confirmed as a vaccine in combination with a boosting agentusing a prime boost protocol. The boosting agent can consist ofrecombinant protein (e.g., Barnett et al., Aids Res. and HumanRetrovinises 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia,for example, expressing a minigene or DNA encoding the complete proteinof interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegahet al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael,Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med.5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boostprotocol is initially evaluated in transgenic mice. In this example,A2.1/K^(b) transgenic mice are immunized IM with 100 μg of a DNAminigene encoding the immunogenic peptides including at least one.HLA-A2 supermotif-bearing peptide. After an incubation period (rangingfrom 3-9 weeks), the mice are boosted IP with 10⁷ pfu/mouse of arecombinant vaccinia virus expressing the same sequence encoded by theDNA minigene. Control mice are immunized with 100 μg of DNA orrecombinant vaccinia without the minigene sequence, or with DNA encodingthe minigene, but without the vaccinia boost. After an additionalincubation period of two weeks, splenocytes from the mice areimmediately assayed for peptide-specific activity in an ELISPOT assay.Additionally, splenocytes are stimulated in vitro with the A2-restrictedpeptide epitopes encoded in the minigene and recombinant vaccinia, thenassayed for peptide-specific activity in an alpha, beta and/or gamma IFNELISA.

It is found that the minigene utilized in a prime-boost protocol elicitsgreater immune responses toward the HLA-A2 supermotif peptides than withDNA alone. Such an analysis can also be performed using HLA-A11 orHLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 orHLA-B7 motif or supermotif epitopes. The use of prime boost protocols inhumans is described below in the Example entitled “Induction of CTLResponses Using a Prime Boost Protocol.”

Example 16 Polyepitopic Vaccine Compositions from Multiple Antigens

The STEAP-1 peptide epitopes of the present invention are used inconjunction with epitopes from other target tumor-associated antigens,to create a vaccine composition that is useful for the prevention ortreatment of cancer that expresses STEAP-1 and such other antigens. Forexample, a vaccine composition can be provided as a single polypeptidethat incorporates multiple epitopes from STEAP-1 as well astumor-associated antigens that are often expressed with a target cancerassociated with STEAP-1 expression, or can be administered as acomposition comprising a cocktail of one or more discrete epitopes.Alternatively, the vaccine can be administered as a minigene constructor as dendrite cells which have been loaded with the peptide epitopes invitro.

Example 17 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response forthe presence of specific antibodies, CTL or HTL directed to STEAP-1.Such an analysis can be performed in a manner described by Ogg et al.,Science 279:2103-2106, 1998. In this Example, peptides in accordancewith the invention are used as a reagent for diagnostic or prognosticpurposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetramericcomplexes (“tetramers”) are used for a cross-sectional analysis of, forexample, STEAP-1 HLA-A*0201-specific CTL frequencies from HLAA*0201-positive individuals at different stages of disease or followingimmunization comprising a STEAP-1 peptide containing an A*0201 motif.Tetrameric complexes are synthesized as described (Musey et al., N.Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201in this example) and β2-microglobulin are synthesized by means of aprokaryotic expression system. The heavy chain is modified by deletionof the transmembrane-cytosolic tail and COOH-terminal addition of asequence containing a BirA enzymatic biotinylation site. The heavychain, β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′ triphosphate and magnesium.Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, andthe tetrameric product is concentrated to 1 mg/ml. The resulting productis referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one millionPBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl ofcold phosphate-buffered saline. Tri-color analysis is performed with thetetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. ThePBMCs are incubated with tetramer and antibodies on ice for 30 to 60 minand then washed twice before formaldehyde fixation. Gates are applied tocontain >99.98% of control samples. Controls for the tetramers includeboth A*0201-negative individuals and A*0201-positive non-diseaseddonors. The percentage of cells stained with the tetramer is thendetermined by flow cytometry. The results indicate the number of cellsin the PBMC sample that contain epitope-restricted CTLs, thereby readilyindicating the extent of immune response to the STEAP-1 epitope, andthus the status of exposure to STEAP-1, or exposure to a vaccine thatelicits a protective or therapeutic response.

Example 18 Induction of Immune Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that usedto confirm the efficacy of a DNA vaccine in transgenic mice, such asdescribed above in the Example entitled “The Plasmid Construct and theDegree to Which It Induces Immunogenicity,” can also be used for theadministration of the vaccine to humans. Such a vaccine regimen caninclude an initial administration of, for example, naked DNA followed bya boost using recombinant virus encoding the vaccine, or recombinantprotein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using anexpression vector, such as that constructed in the Example entitled“Construction of “Minigene” Multi-Epitope DNA Plasmids” in the form ofnaked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also beadministered using a gene gun. Following an incubation period of 3-4weeks, a booster dose is then administered. The booster can berecombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.An alternative recombinant virus, such as an MVA, canarypox, adenovirus,or adeno-associated virus, can also be used for the booster, or thepolyepitopic protein or a mixture of the peptides can be administered.For evaluation of vaccine efficacy, patient blood samples are obtainedbefore immunization as well as at intervals following administration ofthe initial vaccine and booster doses of the vaccine. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of responsesufficient to achieve a therapeutic or protective immunity againstSTEAP-1 is generated.

Example 19 Complementary Polynucleotides

Sequences complementary to the STEAP-1-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring STEAP-1. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06software (National Biosciences) and the coding sequence of STEAP-1. Toinhibit transcription, a complementary oligonucleotide is designed fromthe most unique 5′ sequence and used to prevent promoter binding to thecoding sequence. To inhibit translation, a complementary oligonucleotideis designed to prevent ribosomal binding to a STEAP-1-encodingtranscript.

Example 20 Purification of Naturally-Occurring or Recombinant STEAP-1Using STEAP-1-Specific Antibodies

Naturally occurring or recombinant STEAP-1 is substantially purified byimmunoaffinity chromatography using antibodies specific for STEAP-1. Animmunoaffinity column is constructed by covalently coupling anti-STEAP-1antibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturers instructions.

Media containing STEAP-1 are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of STEAP-1 (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/STEAP-1 binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andGCR.P is collected.

Example 21 Identification of Molecules which Interact with STEAP-1

STEAP-1, or biologically active fragments thereof, are labeled with 1211 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled STEAP-1, washed, and anywells with labeled STEAP-1 complex are assayed. Data obtained usingdifferent concentrations of STEAP-1 are used to calculate values for thenumber, affinity, and association of STEAP-1 with the candidatemolecules.

Example 22 In Vivo Assay for STEAP-1 Tumor Growth Promotion

The effect of the STEAP-1 protein on tumor cell growth is evaluated invivo by evaluating tumor development and growth of cells expressing orlacking STEAP-1. For example, SCID mice are injected subcutaneously oneach flank with 1×10⁶ of either 3T3, or prostate cancer cell lines (e.g.PC3 cells) containing tkNeo empty vector or STEAP-1. At least twostrategies may be used: (1) Constitutive STEAP-1 expression underregulation of a promoter such as a constitutive promoter obtained fromthe genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, provided such promoters are compatible with thehost cell systems, and (2) Regulated expression under control of aninducible vector system, such as ecdysone, tetracycline, etc., providedsuch promoters are compatible with the host cell systems. Tumor volumeis then monitored by caliper measurement at the appearance of palpabletumors and followed over time to determine if STEAP-1-expressing cellsgrow at a faster rate and whether tumors produced by STEAP-1-expressingcells demonstrate characteristics of altered aggressiveness (e.g.enhanced metastasis, vascularization, reduced responsiveness tochemotherapeutic drugs).

Additionally, mice can be implanted with 1×10⁵ of the same cellsorthotopically to determine if STEAP-1 has an effect on local growth inthe prostate, and whether STEAP-1 affects the ability of the cells tometastasize, specifically to lymph nodes, and bone (Miki T et al, OncolRes. 2001; 12:209; Fu X et al, Int J Cancer. 1991, 49:938). The effectof STEAP on bone tumor formation and growth may be assessed by injectingprostate tumor cells intratibially.

The assay is also useful to determine the STEAP-1 inhibitory effect ofcandidate therapeutic compositions, such as for example, STEAP-1intrabodies, STEAP-1 antisense molecules and ribozymes.

Example 23 STEAP-1 Monoclonal Antibody-Mediated Inhibition of Tumors InVivo

The significant expression of STEAP-1 in cancer tissues and surfacelocalization, together with its restrictive expression in normal tissuesmakes STEAP-1 a good target for antibody therapy. Similarly, STEAP-1 isa target for T cell-based immunotherapy. Thus, the therapeutic efficacyof anti-STEAP-1 MAbs in human prostate cancer xenograft mouse models isevaluated by using recombinant cell lines such as PC3-STEAP-1, and3T3-STEAP-1 (see, e.g., Kaighn, M. E., et al., Invest Urol, 1979. 17(1):16-23), as well as human prostate xenograft models such as LAPC 9AD(Saffran et al PNAS 1999, 10:1073-1078).

Antibody efficacy on tumor growth and metastasis formation is studied,e.g., in a mouse orthotopic prostate cancer xenograft models. Theantibodies can be unconjugated, as discussed in this Example, or can beconjugated to a therapeutic modality, as appreciated in the art.Anti-STEAP-1 MAbs inhibit formation of both lung and prostatexenografts. Anti-STEAP-1 MAbs also retard the growth of establishedorthotopic tumors and prolonged survival of tumor-bearing mice. Theseresults indicate the utility of anti-STEAP-1 MAbs in the treatment oflocal and advanced stages prostate cancer. (See, e.g., Saffran, D., etal., PNAS 10:1073-1078 or world wide web URLpnas.org/cgi/doi.10.1073/pnas.051624698).

Administration of the anti-STEAP-1 MAbs led to retardation ofestablished orthotopic tumor growth and inhibition of metastasis todistant sites, resulting in a significant prolongation in the survivalof tumor-bearing mice. These studies indicate that STEAP-1 as anattractive target for immunotherapy and demonstrate the therapeuticpotential of anti-STEAP-1 MAbs for the treatment of local and metastaticprostate cancer. This example demonstrates that unconjugated STEAP-1monoclonal antibodies are effective to inhibit the growth of humanprostate tumor xenografts grown in SCID mice; accordingly a combinationof such efficacious monoclonal antibodies is also effective.

Tumor Inhibition Using Multiple Unconiuqated STEAP-1 MAbs

Materials and Methods

STEAP-1 Monoclonal Antibodies:

Monoclonal antibodies are raised against STEAP-1 as described in theExample entitled “Generation of STEAP-1 Monoclonal Antibodies (MAbs).”The antibodies are characterized by ELISA, Western blot, FACS, andimmunoprecipitation for their capacity to bind STEAP-1. Epitope mappingdata for the anti-STEAP-1 MAbs, as determined by ELISA and Westernanalysis, recognize epitopes on the STEAP-1 protein. Immunohistochemicalanalysis of prostate cancer tissues and cells with these antibodies isperformed.

The monoclonal antibodies are purified from ascites or hybridoma tissueculture supernatants by Protein-G Sepharose chromatography, dialyzedagainst PBS, filter sterilized, and stored at −20° C. Proteindeterminations are performed by a Bradford assay (Bio-Rad, Hercules,Calif.). A therapeutic monoclonal antibody or a cocktail comprising amixture of individual monoclonal antibodies is prepared and used for thetreatment of mice receiving subcutaneous or orthotopic injections ofUM-UC3 and CaLu1 tumor xenografts.

Cell Lines and Xenografts

The prostate cancer cell lines, PC3 and LNCaP cell line as well as thefibroblast line NIH 3T3 (American Type Culture Collection) aremaintained in RPMI and DMEM respectively, supplemented with L-glutamineand 10% FBS.

PC3-STEAP-land 3T3-STEAP-1 cell populations are generated by retroviralgene transfer as described in Hubert, R. S., et al., Proc Natl Acad SciUSA, 1999. 96(25): 14523.

The LAPC-9 xenograft, which expresses a wild-type androgen receptor andproduces prostate-specific antigen (PSA), is passaged in 6- to8-week-old male ICR-severe combined immunodeficient (SCID) mice (TaconicFarms) by s.c. trocar implant (Craft, N., et al., Nat Med. 1999, 5:280).Single-cell suspensions of LAPC-9 tumor cells are prepared as describedin Craft, et al.

Xenograft Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ cancercells mixed at a 1:1 dilution with Matrigel (Collaborative Research) inthe right flank of male SCID mice. To test antibody efficacy on tumorformation, i.e. antibody injections are started on the same day astumor-cell injections. As a control, mice are injected with eitherpurified mouse IgG (ICN) or PBS; or a purified monoclonal antibody thatrecognizes an irrelevant antigen not expressed in human cells. Inpreliminary studies, no difference is found between mouse IgG or PBS ontumor growth. Tumor sizes are determined by caliper measurements, andthe tumor volume is calculated as length×width×height. Mice withSubcutaneous tumors greater than 1.5 cm in diameter are sacrificed.

Orthotopic injections are performed under anesthesia by usingketamine/xylazine. For prostate orthotopic studies, an incision is madethrough the abdomen to expose the prostate and LAPC or PC3 tumor cells(5×10⁵) mixed with Matrigel are injected into the prostate capsule in a10-0 volume. To monitor tumor growth, mice are palpated and blood iscollected on a weekly basis to measure PSA levels. The mice aresegregated into groups for the appropriate treatments, with anti-STEAP-1or control MAbs being injected i.p.

Anti-STEAP-1 MAbs Inhibit Growth of STEAP-1-Expressing Xenograft-CancerTumors

The effect of anti-STEAP-1 MAbs on tumor formation is tested by usingLNCaP and LAPC9 orthotopic models. As compared with the s.c. tumormodel, the orthotopic model, which requires injection of tumor cellsdirectly in the mouse prostate, respectively, results in a local tumorgrowth, development of metastasis in distal sites, deterioration ofmouse health, and subsequent death (Saffran, D., et al., PNAS supra).The features make the orthotopic model more representative of humandisease progression and allowed us to follow the therapeutic effect ofMAbs on clinically relevant end points.

Accordingly, tumor cells are injected into the mouse prostate, and 2days later, the mice are segregated into two groups and treated witheither: a) 200-500 p g, of anti-STEAP-1 Ab, or b) PBS three times perweek for two to five weeks.

A major advantage of the orthotopic cancer models is the ability tostudy the development of metastases. Formation of metastasis in micebearing established orthotopic tumors is studies by IHC analysis on lungsections using an antibody against a tumor-specific cell-surface proteinsuch as anti-CK20 for prostate cancer (Lin S et al, Cancer Detect Prey.2001; 25:202).

Another advantage of xenograft cancer models is the ability to studyneovascularization and angiogenesis. Tumor growth is partly dependent onnew blood vessel development. Although the capillary system anddeveloping blood network is of host origin, the initiation andarchitecture of the neovasculature is regulated by the xenograft tumor(Davidoff A M et al, Clin Cancer Res. 2001; 7:2870; Solesvik O et al,Eur J Cancer Clin Oncol. 1984, 20:1295). The effect of antibody andsmall molecule on neovascularization is studied in accordance withprocedures known in the art, such as by IHC analysis of tumor tissuesand their surrounding microenvironment.

Mice bearing established orthotopic tumors are administered 1000 μginjections of either anti-STEAP-1 MAb or PBS over a 4-week period. Micein both groups are allowed to establish a high tumor burden, to ensure ahigh frequency of metastasis formation in mouse lungs. Mice then arekilled and their bladders, livers, bone and lungs are analyzed for thepresence of tumor cells by IHC analysis. These studies demonstrate abroad anti-tumor efficacy of anti-STEAP-1 antibodies on initiation andprogression of prostate cancer in xenograft mouse models. Anti-STEAP-1antibodies inhibit tumor formation of tumors as well as retarding thegrowth of already established tumors and prolong the survival of treatedmice. Moreover, anti-STEAP-1 MAbs demonstrate a dramatic inhibitoryeffect on the spread of local prostate tumor to distal sites, even inthe presence of a large tumor burden. Thus, anti-STEAP-1 MAbs areefficacious on major clinically relevant end points (tumor growth),prolongation of survival, and health.

Effect of STEAP-1 MAbs on the Growth of Human Prostate Cancer Xenoqraftsin Mice

Male ICR-SCID mice, 5-6 weeks old (Charles River Laboratory, Wilmington,Mass. were used. The mice were maintained in a controlled environmentusing the protocols set forth in the NIH Guide for the Care and Use ofLaboratory Animals. A LAPC-9AD androgen-dependent human prostate cancertumor was used to establish xenograft models. Stock tumors regularlymaintained in SCID mice were sterilely dissected, minced, and digestedusing Pronase (Calbiochem, San Diego, Calif.). Cell suspensionsgenerated were incubated overnight at 37 degrees C. to obtain ahomogeneous single-cell suspension.

STEAP-1 M2/92.30 and M2/120.545 were tested at two different doses of100 μg and 500 μg. PBS and anti-KLH MAb were used as controls. The studycohort consisted of 6 groups with 10 mice in each group. MAbs were dosedIP twice a week for a total of 12 doses, starting the same day as tumorcell injection.

Tumor size was monitored through caliper measurements twice a week. Thelongest dimension (L) and the dimension perpendicular to it (W) weretaken to calculate tumor volume using the formula: W²×L/2. Serum PSAconcentration at treatment day 40 for each animal was measured usingcommercial ELISA kit. The Kruskal-Wallis test and the Mann-Whitney Utest were used to evaluate differences of tumor growth and PSA levelamong groups. All tests were two-sided with a=0.05.

The results of the experiment set forth in FIG. 26 and FIG. 27 show thatSTEAP-1 M2/92.30 and M2/120.545 significantly retard the growth of humanprostate xenograft in a dose-dependent manner.

Example 24 Therapeutic and Diagnostic Use of Anti-STEAP-1 Antibodies inHumans

Anti-STEAP-1 monoclonal antibodies are safely and effectively used fordiagnostic, prophylactic, prognostic and/or therapeutic purposes inhumans. Western blot and immunohistochemical analysis of cancer tissuesand cancer xenografts with anti-STEAP-1 MAb show strong extensivestaining in carcinoma but significantly lower or undetectable levels innormal tissues. Detection of STEAP-1 in carcinoma and in metastaticdisease demonstrates the usefulness of the MAb as a diagnostic and/orprognostic indicator. Anti-STEAP-1 antibodies are therefore used indiagnostic applications such as immunohistochemistry of kidney biopsyspecimens to detect cancer from suspect patients.

As determined by flow cytometry, anti-STEAP-1 MAb specifically binds tocarcinoma cells. Thus, anti-STEAP-1 antibodies are used in diagnosticwhole body imaging applications, such as radioimmunoscintigraphy andradioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res20(2A):925-948 (2000)) for the detection of localized and metastaticcancers that exhibit expression of STEAP-1. Shedding or release of anextracellular domain of STEAP-1 into the extracellular milieu, such asthat seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology27:563-568 (1998)), allows diagnostic detection of STEAP-1 byanti-STEAP-1 antibodies in serum and/or urine samples from suspectpatients.

Anti-STEAP-1 antibodies that specifically bind STEAP-1 are used intherapeutic applications for the treatment of cancers that expressSTEAP-1. Anti-STEAP-1 antibodies are used as an unconjugated modalityand as conjugated form in which the antibodies are attached to one ofvarious therapeutic or imaging modalities well known in the art, such asa prodrugs, enzymes or radioisotopes. In preclinical studies,unconjugated and conjugated anti-STEAP-1 antibodies are tested forefficacy of tumor prevention and growth inhibition in the SCID mousecancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6,(see, e.g., the Example entitled “STEAP-1 Monoclonal Antibody-mediatedInhibition of Bladder and Lung Tumors In Vivo”). Either conjugated andunconjugated anti-STEAP-1 antibodies are used as a therapeutic modalityin human clinical trials either alone or in combination with othertreatments as described in following Examples.

Example 25 Human Clinical Trials for the Treatment and Diagnosis ofHuman Carcinomas Through Use of Human Anti-STEAP-1 Antibodies In Vivo

Antibodies are used in accordance with the present invention whichrecognize an epitope on STEAP-1, and are used in the treatment ofcertain tumors such as those listed in Table I. Based upon a number offactors, including STEAP-1 expression levels, tumors such as thoselisted in Table I are presently preferred indications. In connectionwith each of these indications, three clinical approaches aresuccessfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated withanti-STEAP-1 antibodies in combination with a chemotherapeutic orantineoplastic agent and/or radiation therapy. Primary cancer targets,such as those listed in Table I, are treated under standard protocols bythe addition anti-STEAP-1 antibodies to standard first and second linetherapy. Protocol designs address effectiveness as assessed by reductionin tumor mass as well as the ability to reduce usual doses of standardchemotherapy. These dosage reductions allow additional and/or prolongedtherapy by reducing dose-related toxicity of the chemotherapeutic agent.Anti-STEAP-1 antibodies are utilized in several adjunctive clinicaltrials in combination with the chemotherapeutic or antineoplastic agentsadriamycin (advanced prostrate carcinoma), cisplatin (advanced head andneck and lung carcinomas), taxol (breast cancer), and doxorubicin(preclinical).

II.) Monotherapy: In connection with the use of the anti-STEAP-1antibodies in monotherapy of tumors, the antibodies are administered topatients without a chemotherapeutic or antineoplastic agent. In oneembodiment, monotherapy is conducted clinically in end stage cancerpatients with extensive metastatic disease. Patients show some diseasestabilization. Trials demonstrate an effect in refractory patients withcancerous tumors.

III.) Imaging Agent: Through binding a radionuclide (e.g., iodine oryttrium (I¹³¹, Y⁹⁰) to anti-STEAP-1 antibodies, the radiolabeledantibodies are utilized as a diagnostic and/or imaging agent. In such arole, the labeled antibodies localize to both solid tumors, as well as,metastatic lesions of cells expressing STEAP-1. In connection with theuse of the anti-STEAP-1 antibodies as imaging agents, the antibodies areused as an adjunct to surgical treatment of solid tumors, as both apre-surgical screen as well as a post-operative follow-up to determinewhat tumor remains and/or returns. In one embodiment, a (¹¹¹In) STEAP-1antibody is used as an imaging agent in a Phase I human clinical trialin patients having a carcinoma that expresses STEAP-1 (by analogy see,e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients arefollowed with standard anterior and posterior gamma camera. The resultsindicate that primary lesions and metastatic lesions are identified.

Dose and Route of Administration

As appreciated by those of ordinary skill in the art, dosingconsiderations can be determined through comparison with the analogousproducts that are in the clinic. Thus, anti-STEAP-1 antibodies can beadministered with doses in the range of 5 to 400 mg/m 2, with the lowerdoses used, e.g., in connection with safety studies. The affinity ofanti-STEAP-1 antibodies relative to the affinity of a known antibody forits target is one parameter used by those of skill in the art fordetermining analogous dose regimens. Further, anti-STEAP-1 antibodiesthat are fully human antibodies, as compared to the chimeric antibody,have slower clearance; accordingly, dosing in patients with such fullyhuman anti-STEAP-1 antibodies can be lower, perhaps in the range of 50to 300 mg/m², and still remain efficacious. Dosing in mg/m², as opposedto the conventional measurement of dose in mg/kg, is a measurement basedon surface area and is a convenient dosing measurement that is designedto include patients of all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery ofanti-STEAP-1 antibodies. Conventional intravenous delivery is onestandard delivery technique for many tumors. However, in connection withtumors in the peritoneal cavity, such as tumors of the ovaries, biliaryduct, other ducts, and the like, intraperitoneal administration mayprove favorable for obtaining high dose of antibody at the tumor and toalso minimize antibody clearance. In a similar manner, certain solidtumors possess vasculature that is appropriate for regional perfusion.Regional perfusion allows for a high dose of antibody at the site of atumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops treatments of anti-STEAP-1antibodies in connection with adjunctive therapy, monotherapy, and as animaging agent. Trials initially demonstrate safety and thereafterconfirm efficacy in repeat doses. Trails are open label comparingstandard chemotherapy with standard therapy plus anti-STEAP-1antibodies. As will be appreciated, one criteria that can be utilized inconnection with enrollment of patients is STEAP-1 expression levels intheir tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safetyconcerns are related primarily to (i) cytokine release syndrome, i.e.,hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express STEAP-1.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Anti-STEAP-1 antibodies are found to be safe upon humanadministration.

Example 26 Human Clinical Trial: Monotherapy with Human Anti-STEAP-1Antibody

Anti-STEAP-1 antibodies are safe in connection with the above-discussedadjunctive trial, a Phase II human clinical trial confirms the efficacyand optimum dosing for monotherapy. Such trial is accomplished, andentails the same safety and outcome analyses, to the above-describedadjunctive trial with the exception being that patients do not receivechemotherapy concurrently with the receipt of doses of anti-STEAP-1antibodies.

Example 27 Human Clinical Trial: Diagnostic Imaging with Anti-STEAP-1Antibody

Once again, as the adjunctive therapy discussed above is safe within thesafety criteria discussed above, a human clinical trial is conductedconcerning the use of anti-STEAP-1 antibodies as a diagnostic imagingagent. The protocol is designed in a substantially similar manner tothose described in the art, such as in Divgi et al. J. Natl. CancerInst. 83:97-104 (1991). The antibodies are found to be both safe andefficacious when used as a diagnostic modality.

Example 28 Human Clinical Trial Adjunctive Therapy with HumanAnti-STEAP-1 Antibody and Chemotherapeutic, Radiation, and/or HormoneAblation Therapy

A phase I human clinical trial is initiated to assess the safety of sixintravenous doses of a human anti-STEAP-1 antibody in connection withthe treatment of a solid tumor, e.g., a cancer of a tissue listed inTable I. In the study, the safety of single doses of anti-STEAP-1antibodies when utilized as an adjunctive therapy to an antineoplasticor chemotherapeutic or hormone ablation agent as defined herein, suchas, without limitation: cisplatin, topotecan, doxorubicin, adriamycin,taxol, Lupron, Zoladex, Eulexin, Casodex, Anandron or the like, isassessed. The trial design includes delivery of six single doses of ananti-STEAP-1 antibody with dosage of antibody escalating fromapproximately about 25 mg/m² to about 275 mg/m² over the course of thetreatment in accordance with the following schedule:

Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 MAb Dose 25 75 125 175 225 275mg/m² mg/m² mg/m² mg/m² mg/m² mg/m² Chemotherapy + + + + + + (standarddose)

Patients are closely followed for one-week following each administrationof antibody and chemotherapy. In particular, patients are assessed forthe safety concerns mentioned above: (i) cytokine release syndrome,i.e., hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the human antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express STEAP-1.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Patients are also assessed for clinical outcome, andparticularly reduction in tumor mass as evidenced by MRI or otherimaging.

The anti-STEAP-1 antibodies are demonstrated to be safe and efficacious,Phase II trials confirm the efficacy and refine optimum dosing.

Example 29 Identification and Confirmation of Potential SignalTransduction Pathways

Many mammalian proteins have been reported to interact with signalingmolecules and to participate in regulating signaling pathways. (JNeurochem. 2001; 76:217-223). Fibronectin in particular has beenassociated with the MAPK signaling cascade that control cell mitogenesis(Jiang F, Jia Y, Cohen I. Blood. 2002, 99:3579). In addition, theSTEAP-1 protein contains several phosphorylation sites (see Table XXI)indicating an association with specific signaling cascades. Usingimmunoprecipitation and Western blotting techniques, proteins areidentified that associate with STEAP-1 and mediate signaling events.Several pathways known to play a role in cancer biology can be regulatedby STEAP-1, including phospholipid pathways such as PI3K, AKT, etc,adhesion and migration pathways, including FAK, Rho, Rac-1, Ratenin,etc, as well as mitogenic/survival cascades such as ERK, p38, etc (CellGrowth Differ. 2000, 11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000,19:3003, J. Cell Biol. 1997, 138:913.). In order to determine whetherexpression of STEAP-1 is sufficient to regulate specific signalingpathways not otherwise active in resting PC3 cells, the effect of thesegenes on the activation of the p38 MAPK cascade was investigated in theprostate cancer cell line PC3. Activation of the p38 kinase is dependenton its phosphorylation on tyrosine and serine residues. Phosphorylatedp38 can be distinguished from the non-phosphorylated state by aPhospho-p38 MAb. This phospho-specific Ab was used to study thephosphorylation state of p38 in engineered PC3 cell lines.

PC3 cells were transfected with neomycin resistance gene alone or withSTEAP-1 in pSRa vector. Cells were grown overnight in 0.5% FBS, thenstimulated with 10% FBS for 5 minutes with or without 10 μg/ml MEKinhibitor PD98058. Cell lysates were resolved by 12.5% SDS-PAGE andWestern blotted with anti-phospho-ERK (Cell Signaling) andanti-ERK(Zymed). NIH-3T3 cells were transfected with neomycin resistancegene alone or with STEAP-1 in pSRα vector. Cells were treated as abovebut without the MEK inhibitor. In addition, NIH-3T3-Neo cells weretreated with 10 mg/ml Na salycilate. Expression of STEAP-1 induces thephosphorylation of ERK-1 and ERK-2 in serum and was inhibited by theupstream MEK kinase inhibitor PD98058.

In another set of experiments, the sufficiency of expression of STEAP-1in the prostate cancer cell line PC3 to activate the mitogenic MAPKpathway, namely the ERK cascade, was examined. Activation of ERK isdependent on its phosphorylation on tyrosine and serine residues.Phosphorylated ERK can be distinguished from the non-phosphorylatedstate by a Phospho-ERK MAb. This phospho-specific Ab was used to studythe phosphorylation state of ERK in engineered PC3 cell lines. PC3cells, expressing an activated form of Ras, were used as a positivecontrol.

The results show that while expression of the control neo gene has noeffect on ERK phosphorylation, expression of STEAP-1 in PC3 cells issufficient to induce an increase in ERK phosphorylation (FIG. 28). Theseresults were verified using anti-ERK western blotting and confirm theactivation of the ERK pathway by STEAP-1.

Since FBS contains several components that may contribute toreceptor-mediated ERK activation, we examined the effect of STEAP-1 inlow and optimal levels of FBS. PC3 cells expressing neo or STEAP-1 weregrown in either 0.1% or 10% FBS overnight. The cells were analyzed byanti-Phospho-ERK western blotting. This experiment shows that STEAP-1induces the phosphorylation of ERK in 0.1% FBS, and confirms thatexpression of STEAP-1 is sufficient to induce activation of the ERKsignaling cascade in the absence of additional stimuli.

To confirm that STEAP-1 directly or indirectly activates known signaltransduction pathways in cells, luciferase (luc) based transcriptionalreporter assays are carried out in cells expressing individual genes.These transcriptional reporters contain consensus-binding sites forknown transcription factors that lie downstream of well-characterizedsignal transduction pathways. The reporters and examples of theseassociated transcription factors, signal transduction pathways, andactivation stimuli are listed below.

1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation

3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

4. ARE-luc, androgen receptor; steroids/MAPK;growth/differentiation/apoptosis

5. p53-luc, p53; SAPK; growth/differentiation/apoptosis

6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

7. TCF-luc, TCF/Lef; -catenin, Adhesion/invasion

Gene-mediated effects can be assayed in cells showing mRNA expression.Luciferase reporter plasmids can be introduced by lipid-mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer.

Signaling pathways activated by STEAP-1 are mapped and used for theidentification and validation of therapeutic targets. When STEAP-1 isinvolved in cell signaling, it is used as target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 30 Involvement of STEAP-1 in Small Molecule Transport andCell-Cell Communication

Cell-cell communication is essential in maintaining organ integrity andhomeostasis, both of which become deregulated during tumor formation andprogression. Intercellular communications can be measured using twotypes of assays (J. Biol. Chem. 2000, 275:25207). In the first assay,cells loaded with a fluorescent dye are incubated in the presence ofunlabeled recipient cells and the cell populations are examined underfluorescent microscopy. This qualitative assay measures the exchange ofdye between adjacent cells. In the second assay system, donor andrecipient cell populations are treated as above and quantitativemeasurements of the recipient cell population are performed by FACSanalysis. Using these two assay systems, cells expressing STEAP-1 arecompared to controls that do not express STEAP-1, and it is found thatSTEAP-1 enhances cell communications. FIG. 29 demonstrate that STEAP-1mediates the transfer of the small molecule calcein between adjacentcells, and thereby regulates cell-cell communication in prostate cancercells. In this experiment, recipient PC3 cells were labeled withdextran-Texas Red and donor PC3 cells were labeled with calcein AM(green). The donor (green) and recipient (red) cells were co-cultured at37° C. and analyzed by microscopy for the co-localization of Texas redand calcein. The results demonstrated that while PC3 control cells (nodetectable STEAP-1 protein expression) exhibit little calcein transfer,the expression of STEAP-1 allows the transfer of small molecules betweencells, whereby the initially red recipient cells take on a brownishcolor, and co-localize the red and green molecules. Small moleculesand/or antibodies that modulate cell-cell communication mediated bySTEAP-1 are used as therapeutics for cancers that express STEAP-1. FIG.30 demonstrates that expression of STEAP-1 is necessary on both donorand recipient populations for the transfer of small molecules to takeplace. In this experiment, PC3 cells were transfected with neomycinresistance gene alone or with STEAP-1 in pSRα vector. Recipient cellswere labeled with 1 mg/ml dextran-Texas Red and donor cells were labeledwith 2.5 μg/ml calcein AM. The donor (green) and recipient (red) cellswere co-cultured at 37° C. for 18-24 hours and analyzed by microscopyfor the co-localization of fluorescent dyes. Upper panels: lightmicroscopy; lower panels: UV fluorescence. Left panels: PC3-Neo cellswere both donor and recipient. Center panels: PC3-Neo were donor cellsand PC3-STEAP-1 were recipient. Right panels: PC3-STEAP-1 cells wereboth donor and recipient. Only when STEAP-1 was expressed on both donorand recipient was cell-cell communication detected.

The results show that co-culturing of control PC3 and PC3 cells fail tomediate calcein transfer. Similarly, co-incubation of control PC3 andPC3-STEAP-1 does not allow the transfer of calcein. However,co-culturing PC3-STEAP-1 donor and PC3-STEAP-1 recipient cells mediatessmall molecule transfer as depicted by co-localization of green and redpigments in the same cells. Taken together, the data shown in FIGS. 29and 30 demonstrate that STEAP-1 mediates small molecule transfer andregulates cell-cell communication by forming inter-cellularcommunication channels that are similar in function to gap junctions.

Additionally, STEAP-1 M2/120.545 effect on Gap junction was confirmed(See, FIG. 31). In this experiment, PC3 cells were transfected withneomycin resistance gene alone or with STEAP-1 in pSRα vector. Recipientcells were labeled with 1 mg/ml dextran-Texas Red and donor cells werelabeled with 2.5 μg/ml calcein AM. The donor (green) and recipient (red)cells were co-cultured at 37° C. for 18-24 hours and analyzed bymicroscopy for the co-localization of fluorescent dyes. In allexperiments, the same cells were used as donor and acceptor. Cells wereincubated with the indicated amounts of STEAP-1/120.545 MAb for 10minutes prior to plating and MAb was maintained in the culture for 24hours prior to analysis. STEAP1/120.545 reduces STEAP-1 mediated gapjunction in a dose-dependent manner. The results show thatSTEAP-1/120.545 reduces STEAP-1 mediated gap junction in adose-dependent manner.

Thus, because STEAP-1 functions in cell-cell communication and smallmolecule transport, it is used as a target or marker for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 31 RNA Interference (RNAi)

RNA interference (RNAi) technology is implemented to a variety of cellassays relevant to oncology. RNAi is a post-transcriptional genesilencing mechanism activated by double-stranded RNA (dsRNA). RNAiinduces specific mRNA degradation leading to changes in proteinexpression and subsequently in gene function. In mammalian cells, thesedsRNAs called short interfering RNA (siRNA) have the correct compositionto activate the RNAi pathway targeting for degradation, specificallysome mRNAs. See, Elbashir S. M., et. al., Duplexes of 21-nucleotide RNAsMediate RNA interference in Cultured Mammalian Cells, Nature411(6836):494-8 (2001). Thus, RNAi technology is used successfully inmammalian cells to silence targeted genes.

Loss of cell proliferation control is a hallmark of cancerous cells;thus, assessing the role of STEAP-1 in cell survival/proliferationassays is relevant. Accordingly, RNAi was used to investigate thefunction of the STEAP-1 antigen. To generate siRNA for STEAP-1,algorithms were used that predict oligonucleotides that exhibit thecritical molecular parameters (G:C content, melting temperature, etc.)and have the ability to significantly reduce the expression levels ofthe STEAP-1 protein when introduced into cells. Accordingly, onetargeted sequence for the STEAP-1 siRNA is: 5′ AAGCTCATTCTAGCGGGAAAT 3′(SEQ ID NO: 81). In accordance with this Example, STEAP-1 siRNAcompositions are used that comprise siRNA (double stranded, shortinterfering RNA) that correspond to the nucleic acid ORF sequence of theSTEAP-1 protein or subsequences thereof. Thus, siRNA subsequences areused in this manner are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35 or more than 35 contiguous RNA nucleotides in length. These siRNAsequences are complementary and non-complementary to at least a portionof the mRNA coding sequence. In a preferred embodiment, the subsequencesare 19-25 nucleotides in length, most preferably 21-23 nucleotides inlength. In preferred embodiments, these siRNA achieve knockdown ofSTEAP-1 antigen in cells expressing the protein and have functionaleffects as described below.

The selected siRNA (STEAP-1.b oligo) was tested in numerous cell linesin the survival/proliferation MTS assay (measures cellular metabolicactivity). Tetrazolium-based colorimetric assays (i.e., MTS) detectviable cells exclusively, since living cells are metabolically activeand therefore can reduce tetrazolium salts to colored formazancompounds; dead cells, however do not. Moreover, this STEAP-1.b oligoachieved knockdown of STEAP-1 antigen in cells expressing the proteinand had functional effects as described below using the followingprotocols.

Mammalian siRNA Transfections:

The day before siRNA transfection, the different cell lines were platedin media (RPMI 1640 with 10% FBS w/o antibiotics) at 2×10³ cells/well in80 μl (96 well plate format) for the survival/MTS assay. In parallelwith the STEAP-1 specific siRNA oligo, the following sequences wereincluded in every experiment as controls: a) Mock transfected cells withLipofectamine 2000 (Invitrogen, Carlsbad, Calif.) and annealing buffer(no siRNA); b) Luciferase-4 specific siRNA (targeted sequence:5′-AAGGGACGAAGACGAACACUUCTT-3′) (SEQ ID NO: 82); and, c) Eg5 specificsiRNA (targeted sequence: 5′-AACTGAAGACCTGAAGACAATAA-3′) (SEQ ID NO:83). SiRNAs were used at 10nM and 1 μg/ml Lipofectamine 2000 finalconcentration.

The procedure was as follows: The siRNAs were first diluted in OPTIMEM(serum-free transfection media, Invitrogen) at 0.1 uM μM (10-foldconcentrated) and incubated 5-10 min RT. Lipofectamine 2000 was dilutedat 10 μg/ml (10-fold concentrated) for the total number transfectionsand incubated 5-10 minutes at room temperature (RT). Appropriate amountsof diluted 10-fold concentrated Lipofectamine 2000 were mixed 1:1 withdiluted 10-fold concentrated siRNA and incubated at RT for 20-30″(5-fold concentrated transfection solution). 20 μls of the 5-foldconcentrated transfection solutions were added to the respective samplesand incubated at 37° C. for 96 hours before analysis.

MTS Assay:

The MTS assay is a colorimetric method for determining the number ofviable cells in proliferation, cytotoxicity or chemosensitivity assaysbased on a tetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS(b)] and an electron coupling reagent (phenazineethosulfate; PES). Assays were performed by adding a small amount of theSolution Reagent directly to culture wells, incubating for 1-4 hours andthen recording absorbance at 490 nm with a 96-well plate reader. Thequantity of colored formazan product as measured by the amount of 490 nmabsorbance is directly proportional to the mitochondrial activity and/orthe number of living cells in culture.

In order to address the function of STEAP-1 in cells, STEAP-1 wassilenced by transfecting the endogenously expressing STEAP-1 cell lines.As shown in FIG. 32, ERK-1 and ERK-2 phosphorylation were both inducedby 10% serum, and were inhibited by M2/92.30 MAb and siRNA to STEAP-1.In this experiment, PC3 cells were transfected with neomycin resistancegene alone or with STEAP-1 and MAb in pSHα vector. For RNAi knockdown,PCE-STEAP-1 cells were stably transfected with a pPUR-U6-27-STEAP-1vector containing siRNA to STEAP-1. Cells were starved in 0.1% FBS for18 hours at 37° C., placed on ice for 10 minutes without or with 10μg/ml M2/92.30 MAb, brought to RT for 3 minutes then stimulated with 10%FBS for 5 minutes. Cells were lysed in RIPA buffer, whole cell lysatesresolved by 12.5% SDS-PAGE and proteins detected by Western blotting.Phospho-ERK was detected with rabbit antiserum (Cell Signaling) and ERKwas detected with rabbit anti-ERK (Zymed). STEAP-1 was detected withsheep anti-STEAP-1 and actin was detected with anti-actin MAb (SantaCruz).

Additionally, As shown in FIG. 33, Specific STEAP-1 RNAi stablyexpressed in PC3-STEAP-1 cells reduces the STEAP-1 induced cell-cellcommunication. In this experiment, PC3 cells were transfected withneomycin resistance gene alone or with STEAP-1 in pSRa vector. For RNAiknockdown, PCE-STEAP-1 cells were stably transfected with apPUR-U6-27-STEAP-1 vector containing siRNA to STEAP-1 or an emptyvector. Recipient cells were labeled with 1 mg/ml dextran-Texas Red anddonor cells were labeled with 2.5 μg/ml calcein AM. The donor (green)and recipient (red) cells were co-cultured at 37° C. for 18-24 hours andanalyzed by microscopy for the co-localization of fluorescent dyes. Inall experiments, the same cells were used as donor and acceptor.

Another embodiment of the invention is a method to analyze STEAP-1related cell proliferation is the measurement of DNA synthesis as amarker for proliferation. Labeled DNA precursors (i.e. ³H-Thymidine) areused and their incorporation to DNA is quantified. Incorporation of thelabeled precursor into DNA is directly proportional to the amount ofcell division occurring in the culture. Another method used to measurecell proliferation is performing clonogenic assays. In these assays, adefined number of cells are plated onto the appropriate matrix and thenumber of colonies formed after a period of growth following siRNAtreatment is counted.

In STEAP-1 cancer target validation, complementing the cellsurvival/proliferation analysis with apoptosis and cell cycle profilingstudies are considered. The biochemical hallmark of the apoptoticprocess is genomic DNA fragmentation, an irreversible event that commitsthe cell to die. A method to observe fragmented DNA in cells is theimmunological detection of histone-complexed DNA fragments by animmunoassay (i.e. cell death detection ELISA) which measures theenrichment of histone-complexed DNA fragments (mono- andoligo-nucleosomes) in the cytoplasm of apoptotic cells. This assay doesnot require pre-labeling of the cells and can detect DNA degradation incells that do not proliferate in vitro (i.e. freshly isolated tumorcells).

The most important effector molecules for triggering apoptotic celldeath are caspases. Caspases are proteases that when activated cleavenumerous substrates at the carboxy-terminal site of an aspartate residuemediating very early stages of apoptosis upon activation. All caspasesare synthesized as pro-enzymes and activation involves cleavage ataspartate residues. In particular, caspase 3 seems to play a centralrole in the initiation of cellular events of apoptosis. Assays fordetermination of caspase 3 activation detect early events of apoptosis.Following RNAi treatments, Western blot detection of active caspase 3presence or proteolytic cleavage of products (i.e. PARP) found inapoptotic cells further support an active induction of apoptosis.Because the cellular mechanisms that result in apoptosis are complex,each has its advantages and limitations. Consideration of othercriteria/endpoints such as cellular morphology, chromatin condensation,membrane blebbing, apoptotic bodies help to further support cell deathas apoptotic. Since not all the gene targets that regulate cell growthare anti-apoptotic, the DNA content of permeabilized cells is measuredto obtain the profile of DNA content or cell cycle profile. Nuclei ofapoptotic cells contain less DNA due to the leaking out to the cytoplasm(sub-G1 population). In addition, the use of DNA stains (i.e., propidiumiodide) also differentiate between the different phases of the cellcycle in the cell population due to the presence of different quantitiesof DNA in G0/G1, S and G2/M. In these studies the subpopulations can bequantified.

For the STEAP-1 gene, RNAi studies facilitate the understanding of thecontribution of the gene product in cancer pathways. Such active RNAimolecules have use in identifying assays to screen for MAbs that areactive anti-tumor therapeutics. Further, siRNA are administered astherapeutics to cancer patients for reducing the malignant growth ofseveral cancer types, including those listed in Table 1. When STEAP-1plays a role in cell survival, cell proliferation, tumorigenesis, orapoptosis, it is used as a target for diagnostic, prognostic,preventative and/or therapeutic purposes.

Example 32 Modulation of STEAP-1 Function

Ion transport plays an important role regulating cell growthintracellular permeability, molecular trafficking and signaltransduction (Minke B. Cell Mol Neurobiol. 2001, 21:629; Golovine et al,Am J Physiol Heart Circ Physiol. 2001, 280:H746) these are functionsthat are especially relevant to the neoplastic condition. Cell-cellcommunication regulates homeostasis, cell proliferation and cell death(Evans W H, Martin P E. Mol Membr Biol. 2002 19:121; Carruba G, et al,Ann N Y Acad Sci. 2002, 963:156) these functions too are especiallyrelevant to the neoplastic condition.

Using control cell lines and cell lines expressing STEAP-1, inhibitorsof STEAP-1 function are identified. For example, PC3 and PC3-STEAP-1cells can be incubated in the presence and absence of MAb or smallmolecule inhibitors. The effect of these MAb or small moleculeinhibitors are investigated using the ion flux, cell communication,proliferation and signaling assays described above.

Signal transduction and biological output mediated by transporters canbe modulated through various mechanisms, including inhibition ofreceptor and ligand binding, ion antagonists, protein interactions,regulation of ion and small molecule transport, etc (Tang W et al, FrontBiosci 2002, 7:1583). Using control cell lines and cell lines expressingSTEAP-1, modulators (inhibitors or enhancers) of STEAP-1 function areidentified. For example, PC3 and PC3-STEAP-1 cells are incubated in thepresence and absence of MAb or small molecule modulators. In view of thefunctions of STEAP-1 disclosed herein, modulators that are ion channelblockers used in the context of the present invention include suchcompounds as amlodipine, azulene, dihydropyridines, thianines, nifedine,verapamil and their derivatives (Tanaka Y, Shigenobu K. Cardiovasc DrugRev. 2001, 19:297; Djuric D, Mitrovic V, Jakovljevic V.Arzneimittelforschung. 2002, 52:365; Kourie J I, Wood H B. Prog BiophysMol Biol. 2000; 73:91); and, modulators that are inhibitors of cellcommunication used in the context of the present invention include suchcompounds as beta-glycyrrhetinic acid, retinoids, TPA (Krutovskikh V Aet al, Oncogene. 2002, 21:1989; Rudkin et al, J Surg Res. 2002, 103:183;Ruch J et al, J Cell Biochem. 2001, 83:163). Accordingly, the effect(s)of MAb or small molecule inhibitors are investigated using the ion flux,cell communication, proliferation and signaling assays describedExamples above.

When MAb and small molecules modulate, e.g., inhibit, the transport andtumorigenic function of STEAP-1, they are used for preventative,prognostic, diagnostic and/or therapeutic purposes.

Throughout this application, various website data content, publications,patent applications and patents are referenced. (Websites are referencedby their Uniform Resource Locator, or URL, addresses on the World WideWeb.) The disclosures of each of these references are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

Tables:

TABLE I Tissues that Express STEAP-1 when malignant: Prostate BladderKidney Colon Lung Pancreas Ovary Breast Stomach Rectum Lymphoma

TABLE II Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

TABLE III(a) Amino Acid Substitution Matrix Adapted from the GCGSoftware 9.0 BLOSUM62 amino acid substitution matrix (block substitutionmatrix). The higher the value, the more likely a substitution is foundin related, natural proteins. (See world wide web URLikp.unibe.ch/manual/blosum62.html) A C D E F G H I K L M N P Q R S T V WY . 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4 −2−3 −3 −1 −3 — −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4 −3 1−1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E 6−3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −2 0−2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3 −3−3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2−1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 −1−2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3 −2S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y

TABLE III(b) Original residue Conservative substitution Ala (A) Gly;Ser; Val Arg (R) Lys Asn (N) Gln; His Asp (D) Glu Cys (C) Ser Gln (Q)Asn, His Glu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; ValLeu (L) Ile; Val Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile; Val Phe(F) Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; PheVal (V) Ile; Leu; Ala

TABLE IV HLA Class I/II Motifs/SupermotifsTABLE IV (A): HLA Class I Supermotifs/Motifs POSITION POSITION POSITION2 3 C Terminus (Primary (Primary (Primary SUPERMOTIF Anchor) Anchor)Anchor) A1 TI LVMS FWY A2 LIVM ATQ IV MATL A3 VSMA TLI RK A24 YF WIVLMTFI YWLM B7 P VILF MWYA B27 RHK FYL WMIVA B44 E D FWYLIMVA B58 ATS FWYLIVMA B62 QL IVMP FWY MIVLA MOTIFS A1 TSM Y A1 DE AS Y A2.1 LM VQIAT VLIMAT A3 LMVISATF CGD KYR HFA A11 VTMLISAGN CDF K RYH A24 YF WM FLIWA*3101 MVT ALIS R K A*3301 MVALF IST RK A*6801 AVT MSLI RK B*0702 P LMFWYAIV B*3501 P LMFWY IVA B51 P LIVF WYAM B*5301 P IMFWY ALV B*5401 PATIV LMFWY Bolded residues are preferred, italicized residues are lesspreferred: A peptide is considered motif-bearing if it has primaryanchors at each primary anchor position for a motif or supermotif asspecified in the above table.

TABLE IV (B) HLA Class II Supermotif 1 6 9 W, F, Y, V, .I, L A, V, I, L,P, C, S, T A, V, I, L, C, S, T, M, Y

TABLE IV (C) HLA Class II Motifs MOTIFS 1° anchor 1 2 3 4 5 1° anchor 67 8 9 DR4 preferred FMYLIVW M T W I VSTCPALIM MH MH deleterious R WDEDR1 preferred MFLIVWY PAMQ CW VMATSPLIC M AVM deleterious C CH FD D GDED DR7 preferred MFLIVWY M W A IVMSACTPL M IV deleterious C G GRD N G DR3MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1° anchor 6 Motif a LIVMFY Dpreferred LIVMFAY DNQEST Motif b preferred KRH DR Supermotif MFLIVWYVMSTACPLI Italicized residues indicate less preferred or “tolerated”residues

TABLE IV (D) HLA Class I Supermotifs SUPER- C- MOTIFS POSITION: 1 2 3 45 6 7 8 terminus A1 1° Anchor 1° Anchor TILVMS FWY A2 1° Anchor 1°Anchor LIVMATQ LIVMAT A3 Preferred 1° Anchor YFW YFW YFW P 1° AnchorVSMATLI (4/5) (3/5) (4/5) (4/5) RK deleterious DE (3/5); DE P (5/5)(4/5) A24 1° Anchor 1° Anchor YFWIVLMT FIYWLM B7 Preferred FWY (5/5) 1°Anchor FWY FWY FAnchor LIVM P (4/5) (3/5) VILFMWYA (3/5) deleteriousDE (3/5); DE G QN DE P(5/5); (3/5) (4/5) (4/5) (4/5) G(4/5); A(3/5);QN(3/5) B27 1° Anchor 1° Anchor RHK FYLWMIVA B44 1° Anchor 1° Anchor EDFWYLIMVA B58 1° Anchor 1° Anchor ATS FWYLIVMA B62 1° Anchor 1° AnchorQLIVMP FWYMIVLA Italicized residues indicate less preferred or“tolerated” residues

TABLE IV (E) HLA Class I Motifs 9 Or C- C- POSITION 1 2 3 4 5 6 7 8terminus terminus A1 Preferred GFYW 1° Anchor DEA YFW P DEQ YFW 1°Anchor 9-mer STM N Y deleterious DE RHKLIVMP A G A A1 Preferred GRHKASTCLIVM 1° Anchor GSTC ASTC LIVM DE 1° Anchor 9-mer DEAS Y deleteriousA RHKDEPYFW DE PQN RHK PG GP A1 10- Preferred YFW 1° Anchor DEAQN AYFWQN PAST GDE P 1° Anchor mer STM C A RHK deleterious GP RHKGLIVM DERHK QNA YFW RHK A1 Preferred YFW STCLIVM 1° Anchor A YFW PG G YFW 1°Anchor 10-mer DEAS Y deleterious RHK RHKDEPYFW P G PRHK QN A2.1Preferred YFW 1° Anchor YFW STC YFW A P 1° Anchor 9-mer LMIVQAT DERVLIMAT deleterious DEP DERKH RKH KH A2.1 Preferred AYFW 1° Anchor LVIM GG FYWL 1° Anchor 10-mer LMIVQAT VIM VLIMAT deleterious DEP DE RKHA P RKHDERK HRKH A3 Preferred RHK 1° Anchor YFW PRHKYF A YFW P 1° AnchorLMVISATFCGD W KRYHF deleterious DEP DE A A11 Preferred A 1° Anchor YFWYFW A YFW YFW P 1° Anchor VTLMISAGNCD A KRYH deleterious DEP F G A24Preferred YFWRHK 1° Anchor STC QNP YFW YFW 1° Anchor 9-mer YFWM DER FLIWdeleterious DEG DE G HKG AQN A24 Preferred 1° Anchor P YFWP P 1° Anchor10-mer YFWM FLIW deleterious GDE QN RHK DE A QN DEA A3101 Preferred RHK1° Anchor YFW P YFW YFW AP 1° Anchor MVTALIS ADE DE RK deleterious DEPDE DE DE A3301 Preferred 1° Anchor YFW AYF 1° Anchor MVALFIST W RKdeleterious GP DE A6801 Preferred YFWSTC 1° Anchor YFWLIV YFW P 1°Anchor AVTMSLI DEG M A RK deleterious GP RHK B0702 Preferred RHKFWY 1°Anchor RHK RHK RHK RHK PA 1° Anchor P LMFWY deleterious DEQNP DEP DE DEGDE QN DE AIV A1 Preferred GFYW 1° Anchor DEA YFW P DEQ YFW 1° Anchor9-mer STM N Y deleterious DE RHKLIVMP A G A A1 Preferred GRHK ASTCLIVM1° Anchor GSTC ASTC LIVM DE 1° Anchor 9-mer DEAS Y deleterious ARHKDEPYFW DE PQN RHK PG GP B3501 Preferred FWYLIVM 1° Anchor FWY FWY 1°Anchor P LMFWY deleterious AGP G G IVA B51 Preferred LIVMFWY 1° AnchorFWY STC FWY G FWY 1° Anchor P LIVFWY deleterious AGPDER DEQ GDE AM HKSTCDE G N B5301 Preferred LIVMFWY 1° Anchor FWY STC FWY LIVM FWY 1° AnchorP FWY IMFWY deleterious AGPQN G RHK ALV QN DE B5401 Preferred FWY 1°Anchor FWYLIVM LIVM ALIV FWY 1° Anchor P M AP ATIVLM QND FWY deleteriousGPQNDE GDESTC RHKDE DE GE DE

TABLE IV (F) Summary of HLA-supertypesOverall phenotypic frequencies of HLA-supertypes in different ethnic populationsSpecificity Phenotypic is frequency Supertype Position 2 C-TerminusCaucasian N.A.  Black Japanese Chinese Hispanic Average B7 P AILMVFWY43.2 55.1 57.1 43.0 49.3 49.5 A3 AILMVST RK 37.5 42.1 45.8 52.7 43.144.2 A2 AILMVT AILMVT 45.8 39.0 42.4 45.9 43.0 42.2 A24 YF(WIVLMT)FI (YWLM) 23.9 38.9 58.6 40.1 38.3 40.0 B44 E (D) FWYLIMVA 43.0 21.242.9 39.1 39.0 37.0 A1 TI (LVMS) FWY 47.1 16.1 21.8 14.7 26.3 25.2 B27RHK FYL (WMI) 28.4 26.1 13.3 13.9 35.3 23.4 B62 QL (IVMP) FWY (MIV) 12.64.8 36.5 25.4 11.1 18.1 B58 ATS FWY (LIV) 10.0 25.1 1.6 9.0 5.9 10.3

TABLE IV (G) Calculated population coverage afforded by differentHLA-supertype combinations Phenotypic frequency HLA-supertypes CaucasianN.A Blacks Japanese Chinese Hispanic Average A2, A3 and B7 83.0 86.187.5 88.4 86.3 86.2 A2, A3, B7, A24, B44 99.5 98.1 100.0 99.5 99.4 89.3and A1 99.9 99.6 100.0 99.8 99.9 99.8 A2, A3, B7, A24, B44, A1, B27,B62, and B 58 Motifs indicate the residues defining supertypespecificites. The motifs incorporate residues determined on the basis ofpublished data to be recognized by multiple alleles within thesupertype. Residues within brackets are additional residues alsopredicted to be tolerated by multiple alleles within the supertype.

TABLE IV(h) Frequently Occurring Motifs avrg. % Name identityDescription Potential Function zf-C2H2 34% Zinc finger, C2H2 typeNucleic acid-binding protein functions as transcription factor, nuclearlocation probable cytochrome_b_N 68% Cytochrome b(N- membrane boundoxidase, generate terminal)/b6/petB superoxide Ig 19% Immunoqlobulindomain domains are one hundred amino acids long and include a conservedintradomain disulfide bond. WD40 18% WD domain, G-beta repeat tandemrepeats of about 40 residues, each containing a Trp-Asp motif. Functionin signal transduction and protein interaction PDZ 23% PDZ domain mayfunction in targeting signaling molecules to sub-membranous sites LRR28% Leucine Rich Repeat short sequence motifs involved inprotein-protein interactions Pkinase 23% Protein kinase domain conservedcatalytic core common to both serine/threonine and tyrosine proteinkinases containing an ATP binding site and a catalytic site PH 16% PHdomain pleckstrin homology involved in intracellular signaling or asconstituents of the cytoskeleton EGF 34% EGF-like domain 30-40amino-acid long found in the extracellular domain of membrane- boundproteins or in secreted proteins Rvt 49% Reverse transcriptase(RNA-dependent DNA polymerase) Ank 25% Ank repeat Cytoplasmic protein,associates integral membrane proteins to the cytoskeleton Oxidored_q132% NADH- Ubiquinone/plastoquinone membrane associated. Involved in(complex I), various chains proton translocation across the membraneEfhand 24% EF hand calcium-binding domain, consists of a12 residue loopflanked on both sides by a 12 residue alpha-helical domain Rvp 79%Retroviral aspartyl Aspartyl or acid proteases, centered on protease acatalytic aspartyl residue Collagen 42% Collagen triple helix repeatextracellular structural proteins involved (20 copies) in formation ofconnective tissue. The sequence consists of the G-X-Y and thepolypeptide chains forms a triple helix. Fn3 20% Fibronectin type IIIdomain Located in the extracellular ligand- binding region of receptorsand is about 200 amino acid residues long with two pairs of cysteinesinvolved in disulfide bonds 7tm_1 19% 7 transmembrane receptor sevenhydrophobic transmembrane (rhodopsin family) regions, with theN-terminus located extracellularly while the C-terminus is cytoplasmic.Signal through G proteins

TABLE IV(I) Examples of Medical Isotopes: Isotope Description of useActinium-225 See Thorium-229 (Th-229) (AC-225) Actinium-227 Parent ofRadium-223 (Ra-223) which is an alpha emitter used to treat metastasesin the (AC-227) skeleton resulting from cancer (i.e., breast andprostate cancers), and cancer radioimmunotherapy Bismuth-212 SeeThorium-228 (Th-228) (Bi-212) Bismuth-213 See Thorium-229 (Th-229)(Bi-213) Cadmium-109 Cancer detection (Cd-109) Cobalt-60 Radiationsource for radiotherapy of cancer, for food irradiators, and forsterilization of (Co-60) medical supplies Copper-64 A positron emitterused for cancer therapy and SPECT imaging (Cu-64) Copper-67 Beta/gammaemitter used in cancer radioimmunotherapy and diagnostic studies (i.e.,breast (Cu-67) and colon cancers, and lymphoma) Dysprosium-166 Cancerradioimmunotherapy (Dy-166) Erbium-169 Rheumatoid arthritis treatment,particularly for the small joints associated with fingers and (Er-169)toes Europium-152 Radiation source for food irradiation and forsterilization of medical supplies (Eu-152) Europium-154 Radiation sourcefor food irradiation and for sterilization of medical supplies (Eu-154)Gadolinium-153 Osteoporosis detection and nuclear medical qualityassurance devices (Gd-153) Gold-198 Implant and intracavity therapy ofovarian, prostate, and brain cancers (Au-198) Holmium-166 Multiplemyeloma treatment in targeted skeletal therapy, cancerradioimmunotherapy, bone (Ho-166) marrow ablation, and rheumatoidarthritis treatment Iodine-125 Osteoporosis detection, diagnosticimaging, tracer drugs, brain cancer treatment, (I-125) radiolabeling,tumor imaging, mapping of receptors in the brain, interstitial radiationtherapy, brachytherapy for treatment of prostate cancer, determinationof glomerular filtration rate (GFR), determination of plasma volume,detection of deep vein thrombosis of the legs Iodine-131 Thyroidfunction evaluation, thyroid disease detection, treatment of thyroidcancer as well as (I-131) other non-malignant thyroid diseases (i.e.,Graves disease, goiters, and hyperthyroidism), treatment of leukemia,lymphoma, and other forms of cancer (e.g., breast cancer) usingradioimmunotherapy Iridium-192 Brachytherapy, brain and spinal cordtumor treatment, treatment of blocked arteries (i.e., (Ir-192)arteriosclerosis and restenosis), and implants for breast and prostatetumors Lutetium-177 Cancer radioimmunotherapy and treatment of blockedarteries (i.e., arteriosclerosis and (Lu-177) restenosis) Molybdenum-99Parent of Technetium-99m (Tc-99m) which is used for imaging the brain,liver, lungs, heart, (Mo-99) and other organs. Currently, Tc-99m is themost widely used radioisotope used for diagnostic imaging of variouscancers and diseases involving the brain, heart, liver, lungs; also usedin detection of deep vein thrombosis of the legs Osmium-194 Cancerradioimmunotherapy (Os-194) Palladium-103 Prostate cancer treatment(Pd-103) Platinum-195m Studies on biodistribution and metabolism ofcisplatin, a chemotherapeutic drug (Pt-195m) Phosphorus-32 Polycythemiarubra vera (blood cell disease) and leukemia treatment, bone cancer(P-32) diagnosis/treatment; colon, pancreatic, and liver cancertreatment; radiolabeling nucleic acids for in vitro research, diagnosisof superficial tumors, treatment of blocked arteries (i.e.,arteriosclerosis and restenosis), and intracavity therapy Phosphorus-33Leukemia treatment, bone disease diagnosis/treatment, radiolabeling, andtreatment of (P-33) blocked arteries (i.e., arteriosclerosis andrestenosis) Radium-223 See Actinium-227 (Ac-227) (Ra-223) Rhenium-186Bone cancer pain relief, rheumatoid arthritis treatment and diagnosisand treatment of (Re-186) lymphoma and bone, breast, colon, and livercancers using radioimmunotherapy Rhenium-188 Cancer diagnosis andtreatment using radioimmunotherapy, bone cancer pain relief, (Re-188)treatment of rheumatoid arthritis, and treatment of prostate cancerRhodium-105 Cancer radioimmunotherapy (Rh-105) Samarium-145 Ocularcancer treatment (Sm-145) Samarium-153 Cancer radioimmunotherapy andbone cancer pain relief (Sm-153) Scandium-47 Cancer radioimmunotherapyand bone cancer pain relief (Sc-47) Selenium-75 Radiotracer used inbrain studies, imaging of adrenal cortex by gamma-scintigraphy, lateral(Se-75) locations of steroid secreting tumors, pancreatic scanning,detection of hyperactive parathyroid glands, measure rate of bile acidloss from the endogenous pool Strontium-85 Bone cancer detection andbrain scans (Sr-85) Strontium-89 Bone cancer pain relief, multiplemyeloma treatment, and osteoblastic therapy (Sr-89) Technetium-99m SeeMolybdenum-99 (Mo-99) (Tc-99m) Thorium-228 Parent of Bismuth-212(Bi-212) which is an alpha emitter used in cancer radioimmunotherapy(Th-228) Thorium-229 Parent of Actinium-225 (Ac-225) and grandparent ofBismuth-213 (Bi-213) which are alpha (Th-229) emitters used in cancerradioimmunotherapy Thulium-170 Gamma source for blood irradiators,energy source for implanted medical devices (Tm-170) Tin-117m Cancerimmunotherapy and bone cancer pain relief (Sn-117m) Tungsten-188 Parentfor Rhenium-188 (Re-188) which is used for cancer diagnostics/treatment,bone (W-188) cancer pain relief, rheumatoid arthritis treatment, andtreatment of blocked arteries (i.e., arteriosclerosis and restenosis)Xenon-127 Neuroimaging of brain disorders, high resolution SPECTstudies, pulmonary function tests, (Xe-127) and cerebral blood flowstudies Ytterbium-175 Cancer radioimmunotherapy (Yb-175) Yttrium-90Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver cancertreatment (Y-90) Yttrium-91 A gamma-emitting label for Yttrium-90 (Y-90)which is used for cancer radioimmunotherapy (Y-91) (i.e., lymphoma,breast, colon, kidney, lung, ovarian, prostate, pancreatic, andinoperable liver cancers)

Tables V-XVIII:

Set forth in U.S. patent application Ser. No. 10/236,878; filed 6 Sep.2002, the specific contents are fully incorporated by reference herein.

TABLE XIX Frequently Occurring Motifs avrg. % Name identity DescriptionPotential Function zf-C2H2 34% Zinc finger, C2H2 type Nucleicacid-binding protein functions as transcription factor, nuclear locationprobable cytochrome_b_N 68% Cytochrome b(N-terminal)/b6/petB membranebound oxidase, generate superoxide Ig 19% immunoglobulin domain domainsare one hundred amino acids long and include a conserved intradomaindisulfide bond. WD40 18% WD domain, G-beta repeat tandem repeats ofabout 40 residues, each containing a Trp-Asp motif. Function in signaltransduction and protein interaction PDZ 23% PDZ domain may function intargeting signaling molecules to sub-membranous sites LRR 28% LeucineRich Repeat short sequence motifs involved in protein-proteininteractions Pkinase 23% Protein kinase domain conserved catalytic corecommon to both serine/threonine and tyrosine protein kinases containingan ATP binding site and a catalytic site PH 16% PH domain pleckstrinhomology involved in intracellular signaling or as constituents of thecytoskeleton EGF 34% EGF-like domain 30-40 amino-acid long found in theextracellular domain of membrane-bound proteins or in secreted proteinsRvt 49% Reverse transcriptase (RNA_de- pendent DNA polymerase) Ank 25%Ank repeat cytoplasmic protein, associates integral membrane proteins tothe cytoskeleton Oxidored_q1 32% NADH-Ubiquinone/plastoquinone membraneassociated. Involved in proton (complex I), various chains translocationacross the membrane Efhand 24% EF hand calcium-binding domain, consistsof a12 residue loop flanked on both sides by a 12 residue alpha- helicaldomain Rvp 79% Retroviral aspartyl protease Aspartyl or acid proteases,centered on a catalytic aspartyl residue Collagen 42% Collagen triplehelix repeat (20 extracellular structural proteins involved in copies)formation of connective tissue. The sequence consists of the G-X-Y andthe polypeptide chains forms a triple helix. fn3 20% Fibronectin typeIII domain Located in the extracellular ligand-binding region ofreceptors and is about 200 amino acid residues long with two pairs ofcysteines involved in disulfide bonds 7tm_1 19% 7 transmembrane receptorseven hydrophobic transmembrane regions, with rhodopsin family) theN-terminus located extracellularly while the C-terminus is cytoplasmic.Signal through G proteins

TABLE XX Motifs and Post-translational Modifications of STEAP-1:N-glycosylation site 143 - 146 NGTK (SEQ ID NO: 84)331 - 334 NKTE (SEQ ID NO: 85) Protein kinase C phosphorylation site  3 - 5 SrK 160 - 162 TrK 187 - 189 SyR 246 - 248 TwRCasein kinase II phosphorylation site   3 - 6 SrkD (SEQ ID NO: 86)  8 - 11 TnqE (SEQ ID NO: 87) 240 - 243 SvsD (SEQ ID NO: 88)246 - 249 TwrE (SEQ ID NO: 89) Tyrosine kinase phosphorylation site19 - 27 RRNLEEDDY (SEQ ID NO: 90) N-myristoylation site133 - 138 GVIAAI (SEQ ID NO: 91) 265 - 270 GTIHAL (SEQ ID NO: 92)Bipartite nuclear targeting sequence4 - 20 RKDITNQEELWKMKPRR (SEQ ID NO: 93)

TABLE XXI Protein Characteristics of STEAP-1 Bioinformatic URL (Locatedon the World Wide Web Program at) Outcome ORF ORF finder 1193 bp Proteinlength 339 aa Transmembrane region TM Pred (.ch.embnet.org/) 6 TM at aa73-91, 120-141, 163-181, 218-236, 253-274, 286-304 HMMTop(.enzim.hu/hmmtop/) 6 TM at aa 73-90, 117-139, 164-182, 220-238,257-274, 291-309 Sosui (.genome.ad.jp/SOSui/) 6 TM at aa 70-92, 114-136,163-184, 219-241, 255-273, 292-313 TMHMM (.cbs.dtu.dk/services/TMHMM) 6TM at aa 73-95, 117-139, 164-182, 218-240, 252-274, 289-311 SignalPeptide Signal P (.cbs.dtu.dk/services/SignalP/) potential cleavagebetween aa 136 and 137 pl pl/MW tool (.expasy.ch/tools/) 9.2 plMolecular weight pl/MW tool (.expasy.ch/tools/) 39.8 kD LocalizationPSORT http://psort.nibb.ac.jp/ 60% plama membrane, 40% golgi, 30%endoplasmic reticulum PSORT II http://psort.nibb.ac.jp/ 66% endoplasmicreticulum, 11% mitochondria, 11% plasma membrane Motifs Pfam(.sanger.ac.uk/Pfam/) none Prints (.biochem.ucl.ac.uk/) Transformingprotein P21 ras signature, Fibronectin type III repeat signature Blocks(.blocks.fhcrc.org/) Half-A-TPR repeat, Arsenical pump membrane proteinsignature, M protein repeat

Tables XXII-LII:

Set forth in U.S. patent application Ser. No. 10/236,878; filed 6 Sep.2002, the specific contents are fully incorporated by reference herein.

TABLE LII Search Peptides STEAP 1 Variant 1:nonamers, decamers and 15-mers: aa 1-339 (SEQ ID NO: 94)MESRKDITNQ EELWKMKPRR NLEEDDYLHK DTGETSMLKR PVLLHLHQTA HADEFDCPSE  60LQHTQELFPQ WHLPIKIAAI IASLTFLYTL LREVIHPLAT SHQQYFYKIP ILVINKVLPM 120VSITLLALVY LPGVIAAIVQ LHNGTKYKKF PHWLDKWMLT RKQFGLLSFF FAVLHAIYSL 180SYPMRRSYRY KLLNWAYQQV QQNKEDAWIE HDVWRMEIYV SLGIVGLAIL ALLAVTSIPS 240VSDSLTWREF HYIQSKLGIV SLLLGTIHAL IFAWNKWIDI KQFVWYTPPT FMIAVFLPIV 300VLIFKSILFL PCLRKKILKI RHGWEDVTKI NKTEICSQL                        339Variant 2: 9-mers aa 247-258 (SEQ ID NO: 95) WREFHYIQVNNI10-mers aa 246-258 (SEQ ID NO: 96) TWREFHYIQVNNI15-mers aa 241-258 (SEQ ID NO: 97) VSDSLTWREFHYIQVNNI Variant 3:9-mers aa 247- (SEQ ID NO: 98) WREFHYIQIIHKKSDVPESLWDPCLTRFKGLNLIQS10-mers aa 246- (SEQ ID NO: 99) TWREFHYIQIIHKKSDVPESLWDPCLTRFKGLNLIQS15-mers aa 241- (SEQ ID NO: 100)VSDSLTWREFHYIQIIHKKSDVPESLWDPCLTRFKGLNLIQS Variant 4:9-mers aa 160-176 (SEQ ID NO: 101) RKQFGLLSLFFAVLHAI10-mers aa 159-177 (SEQ ID NO: 102) TRKQFGLLSLFFAVLHAIY15-mers aa 154-182 (SEQ ID NO: 103) DKWMLTRKQFGLLSLFFAVLHAIYSLSYP

TABLE LIII Exon Composition of STEAP-1 (8P1D4) variant 1. Exon numberStart End 1 1 34 2 35 149 3 150 662 4 663 827 5 828 1176

1-44. (canceled)
 45. An isolated polynucleotide encoding a monoclonalantibody that binds to the STEAP-1 protein of SEQ ID NO:3, wherein theantibody comprises all heavy and light chain complementarity determiningregions (CDRs) from the antibody designated X120.545.1.1 (ATCC Accessionnumber PTA-5803).
 46. The isolated polynucleotide of claim 45, whereinthe monoclonal antibody is the monoclonal antibody designatedX120.545.1.1 (ATCC Accession number PTA-5803).
 47. The isolatedpolynucleotide of claim 45, wherein the monoclonal antibody is ahumanized antibody.
 48. The isolated polynucleotide of claim 45, whereinthe monoclonal antibody is an antigen binding antibody fragment.
 49. Theisolated polynucleotide of claim 48, wherein the antibody fragment is anFab, F(ab′)2, Fv or Sfv fragment.
 50. An isolated polynucleotideencoding a monoclonal antibody which is a humanized form of themonoclonal antibody designated X120.545.1.1 (ATCC Accession numberPTA-5803), wherein the monoclonal antibody specifically binds to theSTEAP-1 protein of SEQ ID NO:3.
 51. An isolated polynucleotide encodinga recombinant protein comprising the antigen binding fragment of amonoclonal antibody that binds to the STEAP-1 protein of SEQ ID NO:3,wherein the antibody comprises all heavy and light chain complementaritydetermining regions (CDRs) from the antibody designated X120.545.1.1(ATCC Accession number PTA-5803).
 52. An isolated polynucleotideencoding a recombinant protein comprising the antigen binding fragmentof a monoclonal antibody which is a humanized form of the monoclonalantibody designated X120.545.1.1 (ATCC Accession number PTA-5803),wherein the monoclonal antibody specifically binds to the STEAP-1protein of SEQ ID NO:3.
 53. An isolated vector comprising thepolynucleotide of claim
 45. 54. An isolated vector comprising thepolynucleotide of claim
 50. 55. An isolated vector comprising thepolynucleotide of claim
 51. 56. An isolated vector comprising thepolynucleotide of claim
 52. 57. An isolated host cell comprising thevector of claim
 53. 58. An isolated host cell comprising the vector ofclaim
 54. 59. An isolated host cell comprising the vector of claim 55.60. An isolated host cell comprising the vector of claim 56.